WO2017028782A1 - Application of brain-derived neurotrophic factor precursor protein as target spot for treating affective disorders - Google Patents

Abstract

Application of a binding molecule having an activity neutralizing or inhibiting function on brain-derived neurotrophic factor precursor protein (proBDNF) or a signal transfer molecule thereof, or a binding molecule having an activity stimulating or promoting function on BDNF or a signal transfer molecule thereof, in preparing medicine for preventing, relieving or treating affective disorders. It is found for the first time that proBDNF is an important target spot for treating affective disorders comprising depression disorder, and a binding molecule of the proBDNF is specifically combined, so as to have a significant effect of preventing, relieving or treating affective disorders.

Description

Brain-derived neurotrophic factor precursor protein is used as a target for the treatment of affective disorders Technical field

The present invention belongs to the field of biopharmaceuticals, and particularly relates to a binding molecule having neutralizing or inhibiting activity against a brain-derived neurotrophic factor precursor protein (proBDNF) or a signal transduction molecule thereof, which is prepared for prevention, alleviation or treatment including depression. The application of the drug for affective disorders.

Background technique

Depression is a mental disorder that seriously endangers the physical and mental health of human beings. It seriously affects the daily work and life of patients. Data show that 15% of people with depression are accompanied by suicidal tendencies, and nearly 60% of suicides suffer from depression or other types of affective disorder. According to the World Health Report 2001 published by the World Health Organization, depression is now the fourth largest disease in the world. It is expected that by 2020, depressive disorder will become one of the most serious disease burdens in developing countries, and major depression will be the second leading cause of death and disability.

Adult patients with depression often show low mood, lack of interest, low self-evaluation, inappropriate guilt, despair and self-hatred. Some patients may also have loss of appetite, weight loss, inattention, insomnia, memory. Decline, reduced social activities, decreased sexual desire, etc., and even committed suicide. These symptoms are chronic, but the frequency of relapses gradually increases over time and gradually worsens with each relapse. Although the incidence of depression has increased year by year, its pathogenesis is not very clear. There are many hypotheses about depression. Many researchers believe that depression is affected by genetic factors, social factors, environmental factors, neurotransmitters and their receptor dysfunction, and neuroendocrine axis abnormalities.

In recent years, on the basis of a series of clinical and experimental studies, some scholars have proposed the theory of "neurotrophic and plasticity" of depression. According to the theory, the occurrence of depression is mainly caused by a decrease in the production of neurotrophic factors represented by brain-derived neurotrophic factor (BDNF) in the central nervous system and a decrease in the regeneration of mature hippocampal neurons. It is related to the change of plasticity of hippocampus.

Neurotrophic factors (NTFs) are a class of small molecule polypeptides, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 ( Neurotrophin-3, NT-3) and neurotrophin-4/5 (neurotrophin-4/5, NT-4/5).

BDNF is the second neurotrophic factor discovered after nerve growth factor (NGF) with a molecular weight of 12.4 kDa. It is widely expressed, except for its high expression in the hippocampus and cortex of the central nervous system, and is expressed in the peripheral nervous system, retina, motor neurons, kidneys and prostate, even in human saliva. BDNF.

BDNF contributes to the production and survival of neurons in the central and peripheral nervous systems, and can effectively promote neuronal differentiation and synapse development, increase the density of synaptic terminals and promote the growth of dendrites and axons. Currently in the study of depression, the most involved central nervous system area is the hippocampus. It has been reported in the literature that the hippocampus of depressed patients shrinks compared with the normal population. BDNF is involved in the process of transforming stimuli into synaptic plasticity. It can be seen that BDNF plays an important role in regulating neuronal survival, differentiation, synaptic plasticity and damage repair. There is also evidence that BDNF is not only an important factor in regulating nervous system development and affective disorders, but also an important pain sensation.

Mature BDNF is formed by cleavage of precursor for brain-derived neurotrophic factor (pro-BDNF). Mature BDNF binds to at least two receptors, one is trkB, one of the members of the high-affinity tyrosine kinase family (trk); the other is the low-affinity neurotrophin receptor (LNGFR), also known as p75NTR Receptor. After binding of mature BDNF and trkB, it plays a role in promoting neuronal survival and neuronal axonal elongation through PI3K-Akt, CAMP-PKA, and PKC signaling pathways, and the presence of p75NTR can promote this process, p75NTR The receptor promotes the formation of a high affinity site for the TrkB receptor, thereby enhancing Trk receptor phosphorylation.

In addition to BDNF, the precursor protein proBDNF of BDNF has also received increasing attention. Studies have shown that under normal physiological conditions, pro-BDNF is distributed in the medullary reticular nucleus, solitary tract nucleus, the trigeminal spinal cord and the central canal area, the tail and the ventrolateral medulla, and the ventrolateral ventral ganglia; Dorsal nucleus, dorsal nucleus, trigeminal sensory nucleus, facial nucleus, vestibular cochlear nucleus; gray matter around the midbrain aqueduct, lateral geniculate body; nerve endings in the hippocampus and nerves of the cell body and pyramidal cell layer The nerve endings of the elemental and molecular layers; the neuronal cell bodies and neurite outgrowths of the granule cells of the cerebellum. In addition to the central nervous system, there is also a distribution of proBDNF in the superficial nerve endings of rat peripheral tissues, human saliva, and liver, kidney, and muscle of adult sea otters.

proBDNF is synthesized from the BDNF gene after transcription and translation in the endoplasmic reticulum. The human peptide chain is 247 amino acids in length, the amino acid sequence is SEQ ID NO: 1, and the theoretical molecular weight is 27.8 KD. The molecular weight may range from 32 to 36 kD due to the degree of protein glycosylation modification. The 1-18 position of the molecular amino acid sequence of proBDNF is the signal peptide sequence (SEQ ID NO: 2), which produces two fragments at this position during secretion, one of which contains the amino acids 19-128 of the sequence. The polypeptide fragment of the precursor domain, referred to as the proBDNF pro-domain (SEQ ID NO: 3), and the other fragment is the fragment encoded by the mature domain of amino acid sequence 129-247, after processing Biologically active mature BDNF (SEQ ID NO: 4) was formed. ProBDNF is cleaved intracellularly by Fellin and exosomely cleaved by matrix metalloproteinases (MMPs) and tissue plasminogen activator (tPA) to produce mature BDNF, which in turn promotes neuronal survival and plasticity.

There is currently evidence that proBDNF not only acts as an intermediate for mature BDNF synthesis, but also as a ligand with its high-affinity receptor p75 neurotrophin receptor (p75NTR, SEQ ID NO: 5) and sortilin (SEQ ID NO: 6) Combining to exert biological effects. It is known in the art that neurotrophic factor precursors (including proNGF and proBDNF, etc.) can promote apoptosis and inflammatory responses, but the role of proBDNF in affective disorders is unclear. ProBDNF binds to the extracellular domain of p75NTR and sortilin (SEQ ID NO: 7), forming a complex that transduces several signals including RhoA, JNK and NFKB. ProBDNF triggers neurite collapse by activating RhoA (Sun Y et al, 2012, PlosOne 7(4):e35883).

Summary of invention

The inventors have intensively studied and found for the first time that proBDNF is an important target for the treatment of affective disorders including depression. A binding molecule having a neutralizing or inhibitory activity on a brain-derived neurotrophic factor precursor protein (proBDNF) or a signaling molecule thereof, for example, a binding molecule that specifically binds to proBDNF, particularly a polyclonal antibody against proBDNF, including depression The affective disorder including the disease has a significant mitigating, preventing or treating effect, and thus the present invention has been completed.

The present invention provides the use of a binding molecule having neutralizing or inhibitory activity against a brain-derived neurotrophic factor precursor protein (proBDNF) for the preparation of a medicament for preventing, alleviating or treating a affective disorder.

According to the invention, the affective disorder is selected from the group consisting of depression and anxiety.

According to the present invention, the binding molecule includes a binding molecule that blocks expression of the proBDNF gene, such as antisense RNA, siRNA and miRNA; a binding molecule that specifically binds to proBDNF; or a binding molecule that promotes cleavage of proBDNF into BDNF, such as furin (furin) ), MMP 2, 7, 9 and tissue tissue plasminogen activator (tPA). The binding molecule also includes molecules that bind to the receptor p75 and sortilin or a fragment thereof of proBDNF and block signals triggered by proBDNF, such as compound substances, peptides and antibodies, etc., or miRNA/siRNA, which inhibit p75NTR and sortilin and downstream thereof The expression of molecules.

According to the invention, the binding molecule may also be a molecule that promotes the cleavage of proBDNF into mature BDNF. ProBDNF is known to be a precursor to mature BDNF to those skilled in the art. Mature BDNF is neurotropic, while proBDNF is neurodegenerative. According to the present invention, proBDNF and its receptor are up-regulated in patients with major depression and depression, resulting in an imbalance between the proBDNF signal and the mature BDNF signal. ProBDNF was cleaved by furin, MMP7/9 and tPA. A reasonable way to develop drugs is to find ways to increase proBDNF cuts into mature BDNF drugs, thereby restoring the balance between proBDNF and mature BDNF signals. The drug can be a small chemical or a large protein molecule.

According to the invention, the binding molecule that specifically binds to proBDNF is selected from the group consisting of an antibody p75 (SEQ ID NO: 5), sortilin (SEQ ID NO: 6) or a fragment thereof, such as sortilin ECD, which binds to the precursor protein. -Fc (SEQ ID NO: 8) and p75 ECD-Fc (SEQ ID NO: 9), and functional variants thereof, such as chemically modified variants, substituted, added or deleted variants.

According to the invention, the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a murine antibody or a fragment thereof. Wherein the fragment comprises Fab, F(ab'), F(ab') ₂ , Fv, dAb, Fd, complementarity determining region (CDR) fragment, single chain antibody (scFv), bivalent single chain antibody, single strand Phage antibodies, bispecific diabodies, tri-chain antibodies or four-chain antibodies.

According to the invention, the polyclonal antibody is produced by immunizing an animal with proBDNF or a fragment thereof as an antigen. Wherein the fragment includes, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 10.

The present invention also provides the use of a binding molecule having stimulatory or promoting activity against BDNF or a signaling molecule thereof for restoring BDNF/proBDNF signal balance in the preparation of a medicament for preventing, alleviating or treating affective disorders.

According to the invention, the binding molecule is BDNF or its signaling molecule itself.

According to the invention, the binding molecule is the receptor TrkB of BDNF.

According to the invention, the binding molecule is a sense nucleic acid of BDNF or its receptor TrkB, including sense DNA and sense RNA.

According to the invention, the binding molecule is an agonist of BDNF or TrkB.

Affective disorders are mental disorders characterized by depression, sometimes alternating with manic episodes. Although many people experience sadness or manic emotions from time to time, people with emotional disorders have severe or delayed emotional states that undermine their daily functions. Affective disorders include major depressive disorder, bipolar disorder (bipolar disorder), anxiety and poor mood (depressed personality). In classifying and diagnosing affective disorders, the doctor determines whether the affective disorder is single-phase or biphasic. When the emotional experience has only one extreme (depressed state), the condition is called a single phase. Major depression is a single severe period of depression characterized by negative or desperate thoughts and signs such as exhaustion. In major depression, some patients have separate periods of depression. Some patients do not feel depressed during depression, or have other symptoms associated with depression. Other patients have more frequent periods of depression.

Bipolar depression or bipolar disorder (sometimes called mania) refers to a patient experiencing two emotionally extreme conditions in which the patient alternates between depression (low mood) and mania or mild mania (high mood). . Their emotions went from depression to frenzied and unusually high. Mania is similar to mild mania, but mania is usually more severe and makes patients weak. A bad mood is a recurring or long-term depression that may last a lifetime. A bad mood is similar to major depression, but it is chronic, long-lasting, and mild. The patient's symptoms are not as severe as severe depression, but can last for several years. The mild form of depression seems to always exist. In some cases, the patient also experiences a period of severe depression at a peak of bad mood, sometimes referred to as double depression.

Determining depression using animal models is well known in the art. Many behaviors can be used to test whether an animal experiences depression-like symptoms. These tests include forced swimming test (FST), open field experiment (OFT), elevated plus maze test (EPMT) or sugar water preference test (SPT). FST tests the percentage of time an animal swims or does not swim in the water. OFT tests the distance the animal moves in an open area. EPMT tests whether animals prefer to keep open arms or closed arms. SPT tests whether animals prefer to drink sugar or water. Measurements indicate whether the animal has depression-like symptoms.

The present invention injects proBDNF prepared in insect cells into the lateral compartment of the mouse, resulting in an increase in immobility time in the forced swimming test (FST), indicating that proBDNF can cause depression-like behavior. The invention also proves through a large amount of experimental data that increasing mature BDNF or reducing proBDNF can be slowed down. Solve, prevent or treat depression. Among them, the increase of mature BDNF can be achieved by administering exogenous BDNF or promoting the cutting of proBDNF into BDNF. While reducing proBDNF, there are multiple pathways including, but not limited to, reducing transcription, translation and expression of proBDNF, reducing, neutralizing or inhibiting the biological activity of proBDNF after expression, such as binding of receptors, antibodies or fragments thereof to proBDNF, etc. . Additionally, it will be readily understood by those skilled in the art that downstream signaling molecules that reduce proBDNF can also alleviate, prevent or treat depression. The downstream signaling molecules include, but are not limited to, the receptor p75 and sortilin of proBDNF, or a fragment thereof. Unless otherwise specified, proBDNF, p75 and sortilin or fragments thereof in the present application are collectively referred to as "proBDNF". From the molecular level and the protein level, the binding molecules of the present invention can be classified into nucleic acid agents and protein/antibody agents as agents.

A nucleic acid agent

The binding molecule of the present invention acts as an agent to inhibit gene expression (i.e., inhibit and/or repress gene expression). In the art, the agent is referred to as a "gene silencer", which is well known to those skilled in the art and includes, but is not limited to, nucleic acid sequences, such as RNA, DNA or nucleic acid analogs, which may be single-stranded or double A chain, selected from the group consisting of a nucleic acid encoding a protein of interest, an oligonucleotide, a nucleic acid, a nucleic acid analog, including but not limited to a peptide nucleic acid (PNA), a pseudo-complementary PNA (pc-PNA), a locked nucleic acid (LNA), and derivatives thereof Things and so on. Nucleic acid agents include, but are not limited to, nucleic acid sequences encoding repressor proteins, antisense molecules, ribozymes, small inhibitory nucleic acid molecules including, but not limited to, RNAi, shRNAi, siRNA, microRNAi (miRNA), antisense oligonucleotides, and the like.

In some embodiments, the agent that inhibits proBDNF or a downstream signaling molecule thereof, such as p75 and sortilin, is a nucleic acid. Considering that BDNF is essential for maintaining normal mood, and proBDNF as a precursor of BDNF, it is not advisable to completely block and block the transcription and translation of proBDNF. Therefore, the nucleic acid inhibitor of the present invention is preferably a proBDNF downstream signaling molecule such as a nucleic acid inhibitor of p75 and sortilin or a fragment thereof. Nucleic acid inhibitors include, but are not limited to, RNA interference-inducing molecules such as siRNA, dsRNA, stRNA, shRNA, and variants thereof, wherein the RNA interference molecule silences gene expression of proBDNF. In some embodiments, the nucleic acid inhibitor is an antisense oligo, or a nucleic acid analog, such as DNA, RNA, peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA) or locked nucleic acid (LNA). In an alternative embodiment, the nucleic acid is DNA or RNA, as well as nucleic acid analogs such as PNA, pcPNA and LNA. The nucleic acid may be single-stranded or double-stranded, selected from the group consisting of a nucleic acid encoding a protein of interest, an oligonucleotide, PNA, and the like. Such nucleic acid sequences include, but are not limited to, nucleic acid sequences encoding repressor eggs, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, such as RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, and the like.

In some embodiments, single-stranded RNA (ssRNA), a form of RNA found endogenously in eukaryotic cells, can be used to form RNAi molecules. The ssRNA molecules of the cell include messenger RNA (as well as pre-messeng RNA precursors), small nuclear RNA, small nucleolar RNA, transfer RNA, and ribosomal RNA. Double-stranded RNA (dsRNA) induces a size-dependent immune response such that dsRNA greater than 30 bp activates the interferon response, while shorter dsRNAs will enter the cellular endogenous RNA interference mechanism downstream of the Dicer enzyme.

RNA interference (RNAi) provides a powerful method for inhibiting the expression of selected target polypeptides. RNAi is selectively degraded using small interfering RNA (siRNA) duplexes that target messenger RNA encoding the target polypeptide. siRNA-dependent post-transcriptional silencing of gene expression, including cleavage of target messenger RNA molecules at siRNA-directed sites.

RNA interference (RNAi) is an evolutionarily conserved process that results in the expression or introduction of a sequence of RNA that is identical and highly similar to a target gene, resulting in sequence-specific degradation of messenger RNA (mRNA) transcribed from the targeted gene. Or specific post-transcriptional gene silencing (PTGS) (see Coburn, G. and Cullen, B., (2002) J. of Virology 76(18): 9225), thereby inhibiting expression of the target gene. In one embodiment, the RNA is a double stranded RNA (dsRNA). This process has been described in plants, vertebrate and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes the continued cleavage of long dsRNA into double-stranded fragments called siRNAs. The siRNAs are integrated into a protein complex (referred to as "RNA-induced silencing complex", or "RISC"), which recognizes and cleaves the target mRNA. It can also be introduced by introducing a nucleic acid molecule that inhibits or silences the expression of the target gene. For example, synthetic siRNA or RNA interference agents to initiate RNAi. In this application "Inhibition of target gene expression" is used, including any decrease in expression or protein activity or level of a protein encoded by a target gene or target gene compared to the case where RNAi is not induced. The decrease may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90 compared to the expression of the target gene that has not been targeted by the RNA interference agent or the activity or level of the protein encoded by the target gene. A decrease of %, 95% or 99% or more.

"Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA", is defined as an agent whose function inhibits the expression of a target gene by, for example, RNAi. The siRNA can be chemically synthesized, produced by in vitro transcription, or can be produced in a host cell. In one embodiment, the siRNA is a double stranded RNA (dsRNA) molecule that is from about 15 to about 40 nucleotides in length, preferably from about 15 to about 28 nucleotides, more preferably from about 19 to about 25 nucleotides. The nucleotides are longer, more preferably about 19, 20, 21, 22 or 23 nucleotides in length, and may have 3' and/or 5' overhangs on each strand, which are about 0, 1, 2 in length. 3, 4 or 5 nucleotides. The length of the protruding end is independent between the two chains, i.e. the length of the protruding end of one chain does not depend on the length of the protruding end of the second chain. Preferably, the siRNA is capable of promoting RNA interference by degradation of target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

siRNA also includes small hairpin (also known as stem loop) RNA (shRNA). In one embodiment, these shRNAs comprise a short (eg, about 19 to about 25 nucleotides) antisense strand followed by a nucleotide loop of about 5 to about 9 nucleotides, and a similar sense strand . Optionally, the sense strand can precede the nucleotide loop structure and the antisense strand follow. These shRNAs may be contained in plasmids, retroviruses and lentiviruses and may be expressed, for example, from the pol III U6 promoter or another promoter (see, for example, Stewart et al, RNA; 9(4): 493-501, Apr. 2003 , which is hereby incorporated by reference in its entirety.

The target gene or sequence of the RNA interference agent can be a cellular gene or a genomic sequence, such as a proBDNF sequence. The siRNA can be substantially homologous to the target gene or genomic sequence or a fragment thereof. The term "homologous" as used in this application is defined to be substantially identical, sufficiently complementary or similar to the target mRNA or fragment thereof, capable of performing the target RNA drying Disturb. In addition to natural RNA molecules, RNAs suitable for inhibiting or interfering with expression of a target sequence also include RNA derivatives and analogs. Preferably, the siRNA is identical to its target.

Preferably, the siRNA targets only one sequence. Each RNA interference agent, such as siRNA, can be screened for its potential to reduce the effect of the target by, for example, expression profile analysis. Such methods are known to those skilled in the art and are described, for example, in Jackson et al, Nature Biotechnology 6: 635-637, 2003. In addition to expression analysis, one can also screen similar sequences of potential target sequences in a sequence database to identify potential sequences that can have a reduced target effect. For example, according to Jackson et al. (supra), 15 or perhaps as few as 11 consecutive nucleotide sequences are identical, sufficient to direct silencing of non-targeted transcripts. Therefore, one can screen the proposed siRNAs by using any known sequence alignment methods, such as BLAST for sequence identity analysis, to avoid possible reduction of target silencing effects.

The siRNA molecule is not necessarily limited to a molecule containing only RNA, but, for example, further comprises chemically modified nucleotides and non-nucleotides, and also includes molecules in which the ribose molecule is replaced by another sugar molecule or a molecule having a similar function. . In addition, non-natural bonding between nucleotide residues, such as phosphorothioate linkages, can be used. For example, siRNA containing a D-arabinofuranoside structure in place of the naturally occurring D ribose glycoside found in RNA can be used in the RNAi molecule of the present invention (US Patent Application No. 5,177,196). Other examples include O-bonded RNA molecules between the saccharide and heterocyclic bases of the nucleoside, which confer nuclease resistance and tight complementary strand binding to the oligonucleotide molecule, and contain 2'-O Methylribose, arabinose and especially D-arabinose oligonucleotides are similar (US Patent Application No. 5,177,196).

The RNA strand can be derivatized using a reactive group such as a fluorophore. A particularly useful derivative is a modification at one or more ends of the RNA strand, typically the 3' end of the sense strand. For example, the 2'-hydroxyl group at the 3' end can be easily and selectively derivatized with various groups.

Other useful RNA derivatives include nucleotides having a modified sugar moiety, such as a 2'-O-alkylated residue or a 2'-O-methylribosyl derivative and a 2'-O-fluororibosyl group. derivative. RNA bases can also be modified. Any modified base that can be used to inhibit or interfere with expression of a target sequence can be used. For example, halogenated bases such as 5-bromouracil and 5-iodouracil can be incorporated. The base may also be alkylated, for example, 7-methylguanine may be incorporated in place of the guanine residue. Non-natural bases capable of producing successful inhibition can also be incorporated.

Most preferred siRNA modifications include 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA), as well as RNA duplexes containing phosphodiester bonds or varying amounts of phosphorothioate linkages. Such modifications are known to those skilled in the art and are described, for example, in Braasch et al, Biochemistry, 42: 7967-7975, 2003. Most useful modifications to siRNA molecules can be introduced using chemical methods established by antisense oligonucleotide technology. Preferably, the modification comprises minimal 2'-O-methyl modification, preferably completely free of such modification. Modifications are also preferably free of modification of the free 5'-hydroxyl group of the siRNA.

siRNA and miRNA molecules have various "tails" covalently linked to their 3'- or 5'-ends, as is known in the art. They can be used to stabilize siRNA and miRNA molecules delivered by the methods of the invention. In general, intervening groups attached to the 3' or 5' end of an RNA molecule, various types of reporter groups, and lipophilic groups are well known to those skilled in the art and can be used in the present invention. Methods. 3'-cholesterol or 3'-acridine-modified oligonucleotides can be used to prepare modified RNA molecules useful in the present invention, and a description of their synthesis can be found, for example, in the following article: Gamper, HB, Reed , MW, Cox, T., Virosco, JS, Adams, AD, Gall, A., Scholler, JK and Meyer, RB (1993) Facile Preparation and Exonuclease Stability of 3'-Modified Oligodeoxynucleotides (3'-modified oligodeoxygen nucleus Convenient preparation of nucleotides and exonuclease stability), Nucleic Acids Res. 21145-150; and Reed, MW, Adams, AD, Nelson, JS and Meyer, RB, Jr. (1991) Acridine and Cholesterol-Derivatized Solid Supports for Improved Synthesis of 3'-Modified Oligonucleotides (acridine and cholesterol derivatized solid support for improved synthesis of 3'-modified oligonucleotides), Bioconjugate Chem. 2 217-225 (1993).

Other siRNAs that can be used to target p75NTR and sortilin expression can be easily designed and tested. Thus, siRNAs for use in the methods described herein include siRNA molecules ranging from about 15 to about 40, or from about 15 to about 28 nucleotides in length, which are homologous to the proBDNF gene. Preferably, the siRNA molecule that targets proBDNF is from about 19 to about 25 nucleotides in length. More preferably, the length of the siRNA molecule that targets proBDNF is about 19, 20, 21 or 22 nucleotides. The siRNA molecule that targets proBDNF may also contain a 3' hydroxyl group. siRNA molecules that target proBDNF can be single-stranded or double-stranded; such molecules can be blunt-ended or contain overhanging ends (e.g., 5', 3'). In a specific embodiment, the RNA molecule is double stranded, has blunt ends or contains overhanging ends.

In one embodiment, the RNA molecule that targets at least one strand of p75NTR and sortilin has a 3' overhang of about 0 to about 6 nucleotides in length (e.g., pyrimidine nucleotide, purine nucleotide). In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides in length, from about 1 to about 3 nucleotides, and from about 2 to about 4 nucleotides. In one embodiment, the RNA molecule that targets proBDNF is double-stranded, one strand has a 3' overhang and the other strand can be blunt ended or have overhangs. In embodiments where the RNA molecule that targets proBDNF is double-stranded and both strands contain overhangs, the length of the overhangs of each strand can be the same or different. In a specific embodiment, the RNA of the invention contains about 19, 20, 21 or 22 paired nucleotides and has from about 1 to about 3, especially about 2 nucleosides at the two 3' ends of the RNA. The protruding end of the acid. In one embodiment, the 3' overhang can be stabilized to combat degradation. In a preferred embodiment, the RNA is stabilized by the inclusion of purine nucleotides such as adenine and guanine nucleotides. Optionally, substitution of a pyrimidine nucleotide with a modified analog, such as substitution of a 2'-deoxythymidine nucleoside to a uridine 2 nucleotide 3' overhang, is tolerated and does not affect the potency of RNAi. The absence of a 2' hydroxyl group significantly increases the nuclease resistance of the overhang in tissue culture medium.

The siRNA sequence is selected such that the antisense (guided) strand of the siRNA is maximally taken up into the RISC, thereby maximizing the ability of the RISC to target human p75NTR and sortilin mRNA for degradation. This can be achieved by scanning the sequence with the lowest binding free energy at the 5'-end of the antisense strand. The lower free energy results in increased melting of the 5'-terminus of the siRNA duplex antisense strand, thereby ensuring that the antisense strand will be taken up by RISC and directing sequence-specific cleavage of human p75NTR and sortilin mRNA.

In a preferred embodiment, the siRNA or modified siRNA is delivered in a pharmaceutically acceptable carrier. Other carrier agents such as liposomes can be added to the pharmaceutically acceptable carrier.

In another embodiment, the delivery of the siRNA is by delivery of a vector encoding a small hairpin RNA (shRNA) to a cell in an individual's organ in a pharmaceutically acceptable carrier. After transcription, the shRNA is transformed by the cells into siRNAs capable of targeting, for example, p75NTR and sortilin. In one embodiment, the vector may be a regulatable vector, such as a vector that can be induced with tetracycline.

In one embodiment, the RNA interference agent used in the methods described herein is actively taken up by the cells in vivo after intravenous injection without the use of a carrier, which indicates an RNA interference agent, such as Efficient in vivo delivery of siRNAs used in the methods of the invention.

Other strategies can also be used to deliver RNA interference agents, such as siRNA or shRNA for use in the methods of the invention, such as delivery by a vector, such as a plasmid or viral vector, such as a lentiviral vector. Such vectors which can be used are described, for example, in Xiao-Feng Qin et al, Proc. Natl. Acad. Sci. U.S.A., 100: 183-188. Other methods of delivery include the use of a basic peptide to deliver an RNA interference agent, such as an siRNA or shRNA of the invention, for example by combining or mixing an RNA interference agent with a fragment of a basic peptide, such as a TAT peptide, or by mixing with a cationic lipid. Or formulated in the granules for delivery.

As mentioned, dsRNA, such as siRNA or shRNA, can be used to induce The vector is delivered, for example, a vector that can be induced with tetracycline. The method described in, for example, Wang et al, Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On vectors (using pTet-On vector) (BD Biosciences Clontech, Palo Alto, CA) can be used. In some embodiments, the vector may be a plasmid vector, a viral vector, or any other suitable vector suitable for use in the insertion and exogenous sequences and for introduction into eukaryotic cells. The vector may be an expression vector capable of directing the transcription of the DNA sequence of the agonist or antagonist nucleic acid molecule into RNA. The viral expression vector may be selected, for example, from a retrovirus, a lentivirus, an Epstein Barr virus, a bovine papilloma virus, an adenovirus, and an adeno-associated virus, or a hybrid virus of any of the above viruses. In one embodiment, the vector is extrachromosomal. The use of a suitable extrachromosomal vector provides a means to maintain the antagonizing nucleic acid molecule at a high copy number extrachromosomally in the subject, thereby eliminating the potential effects of chromosomal integration.

RNA interference molecules and nucleic acid inhibitors useful in the methods of the invention can be produced using any known technique, such as direct chemical synthesis, by performing longer double-stranded RNA by exposure to recombinant Dicer protein or Drosophila embryo lysate. Processing, by an in vitro system derived from S2 cells, using bacteriophage RNA polymerase, RNA-dependent RNA polymerase, and DNA-based vectors. The use of cell lysates or in vitro processing may also include subsequent isolation of short, e.g., 21-23 nucleotide siRNAs from lysates or the like. Chemical synthesis is typically carried out by making two single-stranded RNA oligomers and then annealing the two single-stranded RNA oligomers into double-stranded RNA. Other examples include the methods disclosed in WO 99/32619 and WO 01/68836, which teach the chemical and enzymatic synthesis of siRNA. In addition, a large number of commercial service organizations can be used to design and manufacture specific siRNAs (see, for example, QIAGEN Inc., Valencia, CA and AMBION Inc., Austin, TX).

In some embodiments, the agent is a protein or polypeptide or RNAi agent that inhibits expression of p75NTR and sortilin and/or inhibits activity of proBDNF protein. In such embodiments, the cells can be modified (eg, by homologous recombination) to provide increased expression of the agent, such as replacing all or part of a naturally occurring promoter with all or part of a heterologous promoter, such that the cells Higher levels of natural inhibitory agents that express proBDNF, such as proteins or miRNA inhibitors of proBDNF. The heterologous promoter is inserted in such a way that it is operably linked to the target nucleic acid encoding the agent. See, for example, PCT International Publication No. WO 94/12650 to Transkaryotic Therapies, Inc., PCT International Publication No. WO 92/20808 to Cell Genesys, Inc., and PCT International Publication No. WO 91/09955 to Applied Research Systems. The cells can also be engineered to express an endogenous gene containing the agent under the control of an inducible regulatory element, in which case the regulatory sequences of the endogenous gene can be replaced by homologous recombination. The gene activation technique is described in U.S. Patent No. 5,272,071 to Chappel, U.S. Patent No. 5,578,461 to Sherwin et al., PCT/US92/09627 (W093/09222) to Selden et al, and PCT/US90/06436 (WO91/06667) to Skoultchi et al. in. The agent can be prepared by culturing the transformed host cell under culture conditions suitable for expression of the miRNA. The resulting expressed agent can then be purified from such cultures (i.e., from culture media or cell extracts) using known purification techniques such as gel filtration and ion exchange chromatography. Purified nucleic acids may also include an agent inhibitor affinity column containing reagent bound to the protein; one or more column steps over affinity resins, such as concanavalin A- agarose, heparin or -toyopearl ^TM Cibacrom blue 3GA agarose gel; one or more steps involving hydrophobic interaction chromatography, using resins such as phenyl ether, butyl ether or propyl ether; immunoaffinity chromatography, or complementary cDNA affinity chromatography .

In one embodiment, the nucleic acid inhibitor of p75NTR and sortilin can be obtained synthetically, for example by chemical synthesis of the nucleic acid by any synthetic method known to those skilled in the art. The synthetic nucleic acid inhibitors of p75NTR and sortilin can then be purified by any method known in the art. Methods for the chemical synthesis of nucleic acids include, but are not limited to, in vitro chemical synthesis using phosphotriester, phosphate or phosphoramidite chemistry and solid phase techniques, or by deoxynucleoside H-phosphonate intermediates (see Bhongle's) U.S. Patent No. 5,705,629).

In some cases, such as when it is desired to increase nuclease stability, nucleic acids bearing nucleic acid analogs and/or modified internucleoside linkages may be preferred. Nucleic acids containing modified internucleoside linkages can also be synthesized using reagents and methods well known in the art. For example, the synthesis contains a phosphonate, a phosphorothioate, a phosphorodithioate, a phosphoramidate, a methoxyethyl phosphoramidate, a methyl acetal, a thiomethyl acetal, a diisopropylsilane, and a Amide, carbamate, dimethylene sulfide (-CH ₂ -S-CH ₂ ), dimethyl sulfoxide (-CH ₂ -SO-CH ₂ ), dimethyl sulfone (-CH ₂ -SO ₂ - Methods for nucleic acids bonded between CH ₂ ), 2'-O-alkyl and 2'-deoxy-2'-fluorophosphorothioate internucleosides are well known in the art (see Uhlmann et al., 1990, Chem. Rev. 90: 543-584; Schneider et al, 1990, Tetrahedron Lett. 31: 335, and references cited therein). U.S. Patent Nos. 5, 614, 617 and 5, 223, 618 to Acevedo et al., 5, 378, 606 to Acevedo et al., 5, 378, 825 to Cook et al., 5, 672, 697 and 5, 466, 786 to Buhr et al., 5, 777, 092 to Cook et al., 5, 602, 245 to De Mesmacker et al., 5, 610, 289 to Cook et al. Nucleic acid analogs for enhancing nuclease stability and cellular uptake.

Synthetic siRNA molecules, including shRNA molecules, can be obtained using a variety of techniques known to those skilled in the art. For example, siRNA molecules can be chemically synthesized or recombinantly produced using methods known in the art, for example, using appropriately protected ribonucleoside phosphoramidates and conventional DNA/RNA synthesizers (see, for example, Elbashir, SM et al, (2001) Nature. 411: 494-498; Elbashir, SM, W. Lendeckel and T. Tuschl (2001) Genes & Development 15: 188-200; Harborth, J. et al., (2001) J. Cell Science 114: 4557-4565; Masters, XR. (2001) Proc. Natl. Acad. Sci., USA 98: 8012-8017; and Tuschl, T. et al. (1999) Genes & Development 13: 3191-3197). Alternatively, several commercial RNA synthesis suppliers are available, including but not limited to Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (Perbio Science Division, Rockford, IL, USA) ), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, UK). Therefore, siRNA molecules are not very difficult to synthesize and can be easily provided for the quality of RNAi. Furthermore, dsRNA encoded by plasmid vectors, retroviruses and lentiviruses can be expressed in a stem-loop structure (Paddison, PJ et al. (2002) Genes Dev. 16: 948-958; McManus, MT et al. (2002) RNA8 : 842-850; Paul, CP. et al., (2002) Nat. Biotechnol. 20: 505-508; Miyagishi, M. et al., (2002) Nat. Biotechnol. 20: 497-500; Sui, G., et al. 2002) Proc. Natl. Acad. Sci., USA 99: 5515-5520; Brummelkamp, T. et al, (2002) Cancer Cell 2: 243; Lee, NS et al, (2002) Nat. Biotechnol. 20: 500-505; Yu, JY, etc. (2002) Proc. Natl. Acad. Sci., USA 99: 6047-6052; Zeng, Y. et al., (2002) Mol. Cell 9: 1327-1333; Rubinson, DA et al., (2003) Nat. Genet. : 401-406; Stewart, SA, et al. (2003) RNA 9: 493-501). These vectors typically have a polIII promoter upstream of the dsRNA and can express sense and antisense RNA strands respectively and/or as a hairpin structure. In cells, Dicer processes short hairpin RNA (shRNA) into efficient siRNA.

也可用于针对EST文库筛选siRNA序列，以确保只有一个基因被定靶。The targeting region of the siRNA molecule of the invention may be selected from a given target gene sequence, such as the p75NTR and sortilin coding sequences, from about 25 to 50 nucleotides downstream, from about 50 to 75 from the start codon. Nucleotides, or from about 75 to 100 nucleotides. The nucleotide sequence may comprise a 5' or 3' UTR, as well as a region near the initiation codon. One method of designing an siRNA of the invention comprises identifying a 23 nucleotide motif, AA(N19)TT (where N can be any nucleotide), and selecting to have at least 25%, 30%, 35%, 40% A hit sequence of 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. The "TT" portion of the sequence is optional. Alternatively, if such a sequence cannot be found, the search can be extended to use the motif NA (N21), where N can be any nucleotide. In this case, the 3' end of the sense siRNA can be converted to TT to allow for the production of symmetric duplexes relative to the sequence composition of the sense and antisense 3' overhangs. An antisense siRNA molecule can then be synthesized which is complementary to nucleotides 1 to 21 of the 23 nucleotide sequence motif. It may be advantageous to use symmetric 3' TT overhangs to ensure that the formation of small interfering ribonucleoprotein particles (siRNP) contains approximately equal proportions of sense and antisense target RNA cleavage siRNP (Elbashir et al, (2001), supra, and Elbashir et al., 2001, supra). Sequence databases, including but not limited to, analysis of NCBI, BLAST, Derwent, and GenSeq, as well as commercially available oligonucleotide synthesis software such as

It can also be used to screen siRNA sequences for EST libraries to ensure that only one gene is targeted.

Delivery of RNA interference agents: A method of delivering an RNA interference agent, such as siRNA or a vector containing an RNA interference agent, to a target cell, such as a brain cell or other target cell, such as cells of the central and peripheral nervous system, may include (i Injection of a composition comprising an RNA interference agent, such as siRNA, or (ii) direct contact of a cell, such as a brain cell, with a composition comprising an RNA interference agent, such as siRNA. In one embodiment, RNA interference The agent can be targeted to the brain, such as the cortex and hippocampus, where proBDNF is overexpressed. In another embodiment, an RNA interference agent, such as siRNA, can be injected directly into a blood vessel, such as a vein, artery, venule, or arteriole. In another embodiment, the RNA interference agent can be injected directly or applied directly to the affected area.

Administration can be by a single injection or two or more injections. The RNA interference agent is delivered in a pharmaceutically acceptable carrier. One or more RNA interference agents can be used simultaneously. RNA interference agents, such as siRNAs that target p75NTR and/or sortilin mRNA, can be delivered alone or in combination with other RNA interference agents, such as siRNA against other cellular genes. P75NTR and/or sortilin siRNA can also be administered in combination with other agents for treating or preventing affective disorders.

In one embodiment, a particular cell is targeted for RNA interference to limit the potential side effects of RNA interference caused by non-specific targeting of RNA interference. The method may use, for example, a complex or fusion molecule comprising a cell targeting moiety and an RNA interference binding moiety for efficient delivery of RNA interference into the cell. For example, an antibody-protamine fusion protein, when mixed with siRNA, binds siRNA and selectively delivers siRNA to cells expressing the antibody recognized by the antibody, resulting in silencing of gene expression only in cells expressing the antigen. The siRNA or RNA interference inducing molecule binding moiety is a fragment of a protein or nucleic acid binding domain or protein, and the binding moiety is fused to a portion of the targeting moiety. The position of the target moiety can be at the carboxy terminus or amino terminus of the construct, or in the middle of the fusion protein.

siRNA can also be delivered to cells in vitro and in vivo using a virus-mediated delivery mechanism as described in Xia, H. et al. (2002) Nat Biotechnol 20(10): 1006. Plasmid or virus-mediated delivery mechanisms of shRNA can also be used to deliver shRNA to cells in vitro and in vivo, as in Rubinson, DA et al, ((2003) Nat. Genet. 33: 401-406) and Stewart, SA, etc. (2003) RNA 9: 493-501).

RNA interference agents, such as siRNA, can also be introduced into cells via vascular or extravascular circulation, blood or lymphatic systems, and cerebrospinal fluid.

The dose of a particular RNA interference agent is the amount necessary to effect RNA interference of a particular target gene, such as post-translational gene silencing (PTGS), resulting in inhibition of target gene expression or inhibition of the activity or level of the protein encoded by the target gene.

It has also been known that RNAi molecules do not have to exactly match their target sequences. Preferably, however, the 5' and intermediate portions of the siRNA antisense (guide) strand are fully complementary to the target nucleic acid sequence.

Thus, the RNAi formula that functions as a nucleic acid inhibitor of p75NTR and sortilin in the present invention includes, but is not limited to, unmodified and modified double-stranded (ds) RNA molecules, including short-temporal RNA (stRNA) ), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA) (see, eg, Baulcombe, Science 297: 2002-2003, 2002). The dsRNA molecule, e.g., siRNA, may also contain a 3' overhang, preferably a 3' UU or 3' TT overhang. In one embodiment, an siRNA of the invention does not comprise an RNA molecule comprising an ssRNA of more than about 30-40 bases, about 40-50 bases, about 50 bases or more. In one embodiment, the siRNA molecules of the invention have more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90% of their length in their length. of. In some embodiments, the nucleic acid inhibitor of p75NTR and sortilin is any agent that binds to and inhibits expression of proBDNF mRNA, wherein p75NTR and/or sortilin mRNA or its expression (ie, transcription or translation) is inhibited.

In another embodiment of the invention, the agent that inhibits p75NTR and sortilin is a catalytic nucleic acid construct, such as a ribozyme, which is capable of cleaving RNA transcripts, thereby preventing the production of wild-type proteins. Ribozymes use two sequence regions complementary to the target located on either side of the ribozyme catalytic site to target and anneal to a particular sequence. Upon binding, the ribozyme cleaves the target in a site-specific manner. Specific identification and cleavage of sequences of the gene products described herein, such as ribozymes that cleave p75NTR and sortilin or homologs or variants thereof, can be designed and tested by techniques well known to those skilled in the art (eg, Lleber and Strauss, (1995) Mol Cell Biol 15: 540.55l, the disclosure of which is incorporated herein by reference.

In another embodiment of the invention, DNA or RNA encoding mature BDNF or its receptor, such as TrkB, can also be used to treat affective disorders by high expression. The DNA or RNA, also known as a sense nucleic acid, can be injected directly or brought into the body by the cell.

Protein/antibody agent

The binding molecule of the invention is a protein agent. In some embodiments, the agent that inhibits proBDNF and its receptor is a protein and/or peptide inhibitor or fragment thereof, including, but not limited to, a mutated protein, a therapeutic protein, and a recombinant protein. Protein and peptide inhibitors may also include, for example, muteins, genetically modified proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins, and fragments thereof. . In some embodiments, the agent that inhibits proBDNF and its receptor is a dominant negative variant of proBDNF and its receptor, such as a non-functional variant of proBDNF and its receptor.

The binding molecules of the invention may be intact immunoglobulin molecules, such as polyclonal or monoclonal antibodies, or antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab') ₂ , Fv, dAb, Fd, a complementarity determining region (CDR) fragment, a single chain antibody (scFv), a bivalent single chain antibody, a single chain phage antibody, a bispecific diaborating antibody, a triple chain antibody, a four chain antibody, and at least sufficient to confer with proBDNF and A (poly)peptide or a fragment thereof of a fragment of an immunoglobulin that binds to a specific antigen of the receptor. In a preferred embodiment, the binding molecule of the invention is a monoclonal or polyclonal antibody.

In some embodiments, inhibitors of genes and/or gene products useful in the methods of the invention, such as antibodies, include monoclonal, chimeric, humanized, and recombinant antibodies, and antigen-binding fragments thereof. In some embodiments, neutralizing antibodies can be used as inhibitors of proBDNF and its receptors. Antibodies can be readily produced by immunizing an animal, such as a rabbit or mouse, with an antigen. Immunized mice are particularly useful for providing a B cell source for the production of hybridomas that can be cultured to produce large amounts of monoclonal antibodies.

In one embodiment of the invention, the inhibitor of the identified gene product may be an antibody molecule or an epitope binding portion of an antibody molecule or the like. Antibodies provide high binding affinity and unique specificity for a wide range of target antigens and haptens. Monoclonal antibodies for use in the present invention include intact antibodies and fragments thereof which are produced according to conventional techniques, such as hybridoma synthesis, recombinant DNA techniques, and protein synthesis.

Useful monoclonal antibodies and fragments can be derived from any species, including humans, or can be formed as chimeric proteins using sequences from more than one species. Human monoclonal antibodies or "humanized" murine antibodies can also be used in the present invention. For example, a murine monoclonal can be genetically recombined with a nucleotide sequence encoding a murine Fv region (ie, containing an antigen binding site) or its complementarity determining region, and a nucleotide sequence encoding a human constant region domain and an Fc region. Antibodies are "humanized." The humanized targeting moiety is recognized, thereby reducing the immunoreactivity of the antibody or polypeptide in the host receptor, in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2, resulting in increased half-life, adverse immune response The possibility is reduced. Preferably, the murine monoclonal antibody should be used in a humanized form. The antigen binding activity is determined by the sequence and conformation of the amino acids of the six complementarity determining regions (CDRs), which are located in the variable portion (Fv) (three each) of the light and heavy chains of the antibody. 25-kDa single chain Fv (scFv) molecule, a concatenation of the light chain variable region (V _L) and a heavy chain variable region (V _H) connected by a short peptide sequence, is the smallest so far developed antibodies Fragment. Techniques for displaying scFv molecules on the surface of filamentous phage containing the scFv gene have been developed. ScFv molecules with broad antigen specificity may be present in large pools of individual scFv-phage libraries. Some examples of high-affinity monoclonal antibodies and chimeric derivatives thereof that can be used in the methods of the present invention are described in European Patent Application No. 186,833, PCT Patent Application No. WO 92/16,553, and U.S. Patent No. 6,090,923.

A chimeric antibody is an immunoglobulin molecule characterized by two or more fragments or portions derived from different animal species. Generally, the variable region of a chimeric antibody is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Preferably, the two regions and combinations have low immunogenicity when routinely determined.

One limitation of the scFv molecule is the monovalentity of interaction with the target antigen. One of the simplest ways to improve the binding of scFv to its target antigen is to increase its functional affinity by producing multimers. The two, three, and four bodies formed by the combination of the same scFv molecules may contain multiple identical Fv modules. Therefore, these reagents are multivalent, but monospecific. Two different scFv molecules, each containing a V _H and V _L, derived from different parent domains of Ig, they combine to form a fully functional bispecific dimer. A unique application of bispecific scFv is through two (adjacent) surface epitopes that bind simultaneously to two sites of the same target molecule. These reagents have the advantage of a significant increase in affinity compared to a single scFv or Fab fragment. A variety of multivalent scFv-based structures have been engineered, including, for example, miniantibodies, dimeric minibodies, minibodies, (scFv) ₂ , dimers, and trisomies. These molecules have different valences (2 to 4 binding sites), size (50 to 120 kDa), flexibility and ease of production. Single chain Fv antibody fragments (scFvs) mainly monomer, wherein the V _H domains and V _L, connected by a polypeptide linking sequence at least 12 residues. Monomeric scFvs with a 12 and 25 amino acid long ligation sequence are thermodynamically stable under all conditions. Non-covalent dimeric and trimeric molecules can be easily engineered and produced by shortening the peptide linking sequences that link the variable heavy and variable light chains of a single scFv molecule. The scFv dimer is linked by providing a highly flexible amphipathic helix, and the minibody structure can be modified to produce a dimeric bispecific (DiBi) minibody containing two minibodies linked by a double helix (4 scFv molecules). Gene fusion or disulfide-conjugated scFv dimers provide moderate flexibility and can be generated by direct cloning techniques to add C-terminal Gly4Cys sequences. The scFv-CH3 microbody contains two scFv molecules that are linked directly (LD microsomes) or via the very flexible hinge region (Flex microbody) to the IgG CH3 domain. These bivalent constructs have a molecular weight of approximately 80 kDa and are capable of significantly binding antigen. Flex microbodies show impressive tumor localization in mice. Bi- and trispecific multimers can be formed by joining different scFv molecules. When a Fab or a single-chain Fv fragment (scFv) is complexed to form a two-body, three-body or larger aggregate, an increase in functional affinity can be achieved. The most important advantage of multivalent scFvs compared to monovalent scFv and Fab fragments is the increased functional binding affinity (affinity) to the target antigen. The high affinity requires that the scFv multibody be able to bind to different target antigens simultaneously. The increase in functional affinity of the scFv dimer compared to the scFv monomer is significant, mainly due to the decrease in the dissociation rate, which is due to multiple binding to two or more target antigens, and when A recombination caused by a Fv dissociation. When such scFv molecules are joined into multiple bodies, they can be designed to have a high affinity for a single target antigen or multiple specificities for different target antigens. Multiple binding to antigen depends on proper alignment and orientation in the Fv module. For all affinities in a multivalent scFVs target, the antigen binding site must point in the same direction. If multiple binding is not spatially possible, then a significant increase in functional affinity may be due to the increased recombination effect, which depends on the rate of diffusion and antigen concentration. In the present invention, it is also considered to combine antibodies with moieties that improve their properties. For example, combinations of antibodies with PEG that increase their in vivo half-life can be used in the present invention. An immunological library was prepared by PCR amplification of a gene encoding a variable antibody fragment from B lymphocytes that were not exposed to antigen or immunized animals or patients. A combination of oligonucleotides specific for an immunoglobulin gene or an immunoglobulin gene family is used. The immunoglobulin germline gene can be used to make all components of a semi-synthetic antibody with a complementarity determining region of a variable fragment amplified by PCR using degenerate primers. An advantage of these single-part libraries is that antibody fragments directed against a large number of antigens can be isolated from a single library. Phage display technology can be used to increase the affinity of antibody fragments, from existing antibody fragments, by random, codon-based or site-directed mutagenesis, by passing each domain with fragments from all components of the untouched antigen. The domain was shuffled or a new library was prepared by using bacterial strains.

Optionally, SCID-hu mice, such as model mice developed by Genpharm, can be used to produce antibodies or fragments thereof. In one embodiment, a novel high affinity binding molecule called peptabody produced by utilizing multivalent interaction effects is contemplated. The short peptide ligand is fused to the helical coiled component domain of the cartilage oligomeric matrix protein through a semi-rigid hinge region to produce a pentameric multivalent binding molecule. In a preferred embodiment of the invention, a ligand and/or a chimeric inhibitor can be targeted to the tissue by the use of a bispecific antibody, for example by chemically linking an anti-ligand antibody (Ab) to an A-specific target. And the bispecific antibody produced. To avoid the limitations of chemical binding, molecular conjugates of antibodies can be used to produce recombinant bispecific single chain Ab and/or chimeric inhibitors against ligands on cell surface molecules. Optionally, two or more active drugs linked to the targeting moiety can be administered And/or inhibitors, wherein each conjugate comprises a targeting moiety, such as a different antibody. Each antibody reacts with a different target site epitope (associated with the same or a different target site antigen). Different antibodies and agents attached thereto are additionally accumulated at the target site of interest. An antibody-based or non-antibody-based targeting moiety can be used to deliver a ligand or inhibitor to a target site. Preferably, a natural binding agent of an unregulated or disease associated antigen is used for this purpose.

The invention also includes functional variants of the binding molecules. A variant molecule is a functional variant of a binding molecule of the invention, as long as the variant competes with the parental binding molecule for specific binding to proBDNF or a fragment thereof. In other words, the functional variant is still capable of binding to proBDNF or a fragment thereof. Functional variants include, but are not limited to, primary structural sequences that are substantially similar, but contain derivatives such as in vitro or in vivo chemical and/or biochemical modifications not found in the parent binding molecule. Such modifications include acetamylation, deuteration, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxyl groups , methylation, oxidation, PEGylation, proteolytic processing, phosphorylation, etc., as long as the modifications in the amino acid and/or nucleotide sequence of the parent binding molecule are not significantly affected or altered by the nucleotide sequence The binding property of the binding molecule or the amino acid sequence, that is, the binding molecule can still recognize and bind to its target site.

The functional variants can have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can include both natural as well as non-natural nucleotides and amino acids.

Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar structure or chemical nature. Amino acid residues having similar side chains are well known in the art and include amino acids having a basic side chain (e.g., lysine, arginine, histidine), acidic side chain amino acids (e.g., aspartic acid, glutamine) Acid), uncharged polar side chain amino acids (eg, aspartame, glutamine, serine, threonine, tyrosine, hemi-amino acid, tryptophan), non-polar side chain amino acids (eg Glycine, alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine), branched side chain amino acids (eg threonine, Proline, isoleucine) and aromatic side chain amino acids (eg tyrosine, phenylalanine, tryptophan). Those skilled in the art will appreciate that other amino acids than the above amino acids can also be used. In addition, variants may have non-conservative amino acid substitutions, such as amino acid substitutions by another amino acid residue having a different structure or chemical nature. Similar small variations can also include amino acid deletions or insertions, or both. The amino acid residues can be found and determined using computer programs well known in the art to be substituted, inserted or deleted without eliminating immunological activity.

In addition, the functional variants also comprise truncations of the amino acid sequence at the amino terminus or the carboxy terminus or at both ends. Functional variants of the invention may have increased or decreased binding affinity for proBDNF or fragments thereof as compared to the parent binding molecule. Functional variants of the invention have from about 50% to about 99%, preferably from about 60% to about 99%, more preferably from about 70% to about 99%, even more preferably from about 80% to about 99%, of the parent binding molecule. Most preferably from about 90% to about 99%, especially from about 95% to about 99%, and especially from about 97% to about 99%, of amino acid sequence homology. Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally sequence amino acid sequences for comparison and to identify similar or identical amino acid residues. Functional variants can be obtained by altering the parental binding molecule or a portion thereof using common molecular biology methods known in the art including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and Heavy chain and / or light chain shuffling method. In one embodiment, a functional variant of the invention has neutralizing activity for proBDNF. The neutralizing activity can be the same or higher or lower than the parent binding molecule. When the term (human) binding molecule is used, it also encompasses functional variants of the (human) binding molecule.

As a preferred mode of the invention, the binding molecule is a monoclonal antibody. The monoclonal antibodies or fragments thereof of the invention may be humanized, chimeric or murine. As used herein, "humanized antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human, and/or an antibody prepared by techniques known in the art for preparing a humanized antibody. Humanized antibodies mainly refer to murine (or other non-human) monoclonal antibodies modified by gene cloning and DNA recombination technology, and the re-expressed antibodies have most of the amino acid sequences substituted by human sequences, and substantially retain the parental mouse monoclonal antibodies. Affinity and specificity, which reduces its heterogeneity and is beneficial Applied to the human body. Humanized antibodies include chimeric antibodies, modified antibodies (also known as CDR-implanted antibodies), surface remodeling antibodies, or fully humanized antibodies. Humanized antibodies can be produced using a variety of methods known in the art, for example, a humanized antibody is selected from a phage library, wherein the phage library expresses a human antibody. Humanized antibodies can also be prepared by introducing human immunoglobulin sites in transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. In addition, humanized antibodies can also be prepared by immortalizing human B lymphocytes that produce antibodies against a particular antigen.

As a preferred mode of the present invention, the binding molecule is a polyclonal antibody that specifically binds to proBDNF. In the present invention, the term "polyclonal antibody (polyclonal antibody)" refers to a group of globulins which have specific binding ability to an antigen, which is stimulated by an antigen to produce an immunological reaction, which is synthesized by the plasma cells of the body and Secreted. An antigen is usually composed of a plurality of antigenic determinants, and an antigenic determinant stimulates the body, and an antibody produced by receiving a B lymphocyte is called a monoclonal antibody. The body is stimulated by a variety of antigenic determinants, and a variety of monoclonal antibodies are produced accordingly. These monoclonal antibodies are intermixed with polyclonal antibodies. The advantages of polyclonal antibodies are their high potency, high specificity, strong affinity, good sensitivity, and easy handling and quality control. In addition, polyclonal antibody preparation is relatively easy and more economical.

Polyclonal antibodies can be made by a variety of methods well known to those skilled in the art. The proBDNF or a fragment thereof can be administered to an animal (such as sheep, rabbit, mouse, rat, etc.) to induce production of polyclonal antibodies. Similarly, cells expressing proBDNF or fragments thereof can also be used to immunize animals to produce antibodies. Polyclonal antibodies can be prepared by lymph node injection, subcutaneous multiple injection, multi-channel combined injection and other immunization methods. In a specific embodiment of the present invention, a proBDNF fragment (for example, SEQ ID NO: 10) is used as an antigen, and after mixing with Freund's adjuvant, a multi-point injection of the back subcutaneously, immunizing the sheep, and boosting the immunity, finally obtaining high titer. Polyclonal antibody.

3. ProBDNF antagonists and BDNF agonists

ProBDNF binds to stotilin and p75, forming a complex that activates several intracellular signals, such as RhoA, JNK and NFκB. ProBDNF-transduced signals play a key role in triggering neurodegenerative pathways leading to affective disorders. In the field of drug development, it is preferred to employ an antagonist to block the signaling pathway that causes the disease. Since the proBDNF/sortilin/p75 signaling pathway is critical for the development of affective disorders, it is desirable to use antagonists as agents to block signaling pathways.

The agent may be a small molecule that interacts with a binding site between proBDNF, sortilin and p75. The crystallography of ProBDNF, sortilin, and p75 helps to design the drug in a rational manner. Leading drugs can be generated using downstream signals as readout screening drugs.

The agent may also be a macromolecule, such as miRNAs, siRNAs against p75 and sortilin, which reduce the expression of p75 and sortilin such that the signaling pathway leading to the disease for therapeutic purposes is blocked.

The agent may also be a monoclonal antibody or a polyclonal antibody directed against different variants of sortilin and/or p75, preventing the binding of proBDNF to these receptors. The antibody will block the signal of proBDNF and thus have a therapeutic effect on affective disorders.

Further, the agent of the present invention includes an agonist of BDNF or a receptor thereof to stimulate or promote the activity of BDNF or its receptor, restore the balance of BDNF/probDNF signaling, thereby preventing or treating an affective disorder including depression.

4. Pharmaceutical composition

The invention provides a pharmaceutical composition comprising a therapeutically effective amount of the binding molecule. The composition may also comprise a pharmaceutically acceptable carrier. The pharmaceutical compositions can be administered by conventional routes including, but not limited to, intravenous, intraperitoneal injection, and the like. The pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents for depression.

The term "pharmaceutically acceptable carrier" as used in the present invention means that when the binding molecule body and the carrier are combined and appropriately administered to an animal or a human, no adverse, allergic or other adverse reaction occurs. As used herein, a "pharmaceutically acceptable carrier" should be associated with a binding molecule of the invention. It can be blended with it without greatly reducing the effect of the composition under normal circumstances.

By "effective amount" as used herein is meant an amount sufficient to produce a beneficial and desired result, including clinical outcomes that alleviate disease progression or cure. An "effective amount" can be achieved by one or more administrations. The specific dose should be determined by factors such as the route of administration, the condition of the patient, and the like.

润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。Specific examples of some substances as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil , olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as

Wetting agents, such as sodium lauryl sulfate; colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers.

The composition of the present invention can be formulated into various dosage forms according to various needs, and can be administered by a physician according to factors such as patient type, age, body weight, and general disease state, mode of administration, and the like. The mode of administration may be by injection or other treatment.

The binding molecules of the invention may be used in unseparated or isolated form. Furthermore, the binding molecules of the invention may be administered alone or in a mixture comprising at least one binding molecule of the invention (or a variant or fragment thereof). In other words, the binding molecules can be administered in combination, for example as a pharmaceutical composition comprising two or more binding molecules, variants or fragments thereof of the invention. For example, binding molecules having different but complementary activities can be combined in one therapeutic regimen to achieve the desired prophylactic, ameliorating or therapeutic effect, or binding molecules having the same activity can be combined in a single therapeutic regimen to achieve the desired prophylaxis, Relieve or treat.

The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). suitable The dosage range is, for example, from 0.1 to 100 mg/kg body weight, preferably from 0.5 to 15 mg/kg body weight. In addition, for example, one bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased proportionally depending on the urgency of the treatment situation. The binding molecules and compositions of the invention are preferably sterile. Methods for making these molecules and compositions sterile are well known in the art.

V. The present invention successfully built a rat depression model using a chronic unpredictable mild stress (CUMS) method.

1. Selection and grouping of experimental animals

100 adult male Wistar rats, weighing about 200-250 g, were randomly divided into 10 groups. One group was fed normally, fed food and water freely as a control group; the remaining nine groups were given CUMS.

2. Modeling

Rats in the experimental group were housed in single cages and stimulated with CUMS. The rats in the control group were given one cage every 5 animals and fed normally.

CUMS depression model making program:

(1) The cage is tilted 45°, 7 hours

(2) 1 cm from the tail of the rat, 1 minute at the end of the clip

(3) fasting, 24 hours

(4) Water ban, 24 hours

(5) 4 ° C cold water swimming, 5 minutes

(6) Black and white upside down, 24 hours

(7) 200 rev / min horizontal shaking, 5 minutes

(8) Different noise, 6 hours

(9) Put foreign matter, 12 hours

(10) moist padding, 12 hours

(11) Do not give any stimulation, 24 hours

(12) 40 ° C hot water swimming, 5 minutes

The above 12 kinds of stimuli were randomly arranged within 21 days, one type per day, and each stimuli could not appear more than three times. The same kind of stimuli could not appear continuously, and the animals could not predict the occurrence of stimuli. 22nd Behavioral evaluation of all animals: Open-field Test, Forced-swimming Test and 1% Sucrose preference Test. Data before and after treatment. The difference in learning (p<0.05) indicates that the model was established successfully.

6. The present invention uses the following behavioral experimental methods to evaluate a rat depression model and its therapeutic effect (Yang CR et al., 2014 Neurotoxicity Research Neurotox Res. 2014 Apr; 25(3): 235-47; Ruan CS et al., Eur J Neurosci.2014Aug;40(4):2680-90)

(1) 1% sucrose preference test (SPT) determination

1% sugar preference = 1% sugar consumption / (water consumption + 1% sugar consumption)

Before the start of CUMS stimulation, all experimental rats should be trained in syrup adaptation for about 4 days. After 2 bottles of syrup were placed in the cage for 24 hours, one bottle of syrup was replaced with pure water for the same 24 hours, and then banned. The food was banned for 24 hours. Finally, the single rats were single-cage. Each bottle was placed with a bottle of about 75 ml of pure water and a bottle of about 75 ml of 1% syrup for free drinking for 1 hour. After the end of the adaptation phase, 21 days of CUMS stimulation was performed. On the 22nd day, the water was forbidden and fasted for 23 hours. At the 24th hour, the amount of the 1% sucrose solution was measured for 1 hour. The difference in weight of the water bottle before and after the measurement was the amount of 1% sucrose solution consumed by the animal for 1 hour. The 1% sucrose solution consumption was assessed as 1% sucrose consumption/(sum of water and sugar consumption) and was measured once a week. Compared with the control group, the ratio decreased significantly in the model rat group, indicating that the depression in the depression group was decreased, which is one of the core manifestations of depression.

(2) Open-field test (OFT)

METHODS: The animals were placed in a box with a wall of 100 cm × 100 cm × 50 cm and the bottom of the box was black, and the bottom surface was composed of 25 equal 20 cm × 20 cm squares. The video tracking analysis software (ANY-maze) system was automatically divided. The animals were placed in the positive central grid and the measurement was started. Each test was performed for 5 minutes. The test procedure was recorded by a camera. Each rat was only tested once for behavior. After the measurement was completed, the stool was cleaned and the bottom of the box was cleaned with 75% alcohol. The next measurement was performed using a single blind method. The measurement index is the non-exercise time within 5 minutes, the number of climbs (two front paws off the bottom) and the horizontal movement distance (four claws pass through the grid number).

(3) Forced swimming test (FST)

The forced swimming experiment is based on the experimental method established by Porsoltetal. During the experiment, the rats were placed in an open cylindrical white plastic container (40 cm in diameter and 80 cm in height), and 40 cm of water was injected thereto at a temperature of about 22 ° C to 25 ° C. A series of parameters during the desperate, inactive state of the rats in this environment were recorded. The total recording time is 5 min, using a single blind method. The measurement index is the immobility time (the rat floats on the surface of the water with only slight activity or the body is perpendicular to the water surface only when the nose is out of the water). This value was significantly decreased in the model rat group relative to the control group.

7. The present invention uses the following molecular biological methods to detect the transcription and expression levels of BDNF and proBDNF (Zhou L et al: J Affect Disord. 2013 Sep 25; 150(3): 776-84; Xiong J et al., Neuro Oncol. 2013Aug; 15(8): 990-1007)

1. Animal model and related experimental methods

Direct material: Rats were killed by neck dissection: first anesthetize with ether, then cut the mouse head along the neck with scissors, immediately place it on the ice, quickly peel off the scalp, cut the skull, separate the meninges, remove the whole brain, and remove the cerebellum. The brain was removed from the hippocampus and cortex, and it was quickly placed in liquid nitrogen for specimens for western blot and RT-PCR.

Infusion: Rats were anesthetized with 0.3% sodium pentobarbital, the thoracic cavity was opened, the heart was fully exposed, and the perfusion needle was inserted into the left apex in the direction of the aorta. The sterilized 0.9% saline was perfused until the liver. Whitening, and then replaced with 4% paraformaldehyde solution, see muscle twitching, indicating good perfusion, until the body is stiff, the head, body, tail are in line, quickly remove the intact brain tissue. Then put it in 4% paraformaldehyde solution, place it at 4 °C for 12-48 hours, then put it into 30% sucrose solution, place it at 4 °C until the brain tissue sinks, take it out for frozen section (slice 16μm, drift Sheet 30 μm). Keep the tissue for Golgi staining.

2.Western blot

2.1 Protein sample preparation:

Extraction of total protein from tissues:

Place 0.1 g of tissue in the globular part of the homogenizer and cut it as much as possible with clean scissors. Thereafter, 1 ml of RIPA lysate was added to the homogenizer and rapidly homogenized on ice. Place on ice for 10-20 min, pipette the lysate into a 1.5 ml centrifuge tube, centrifuge at 12000 rpm for 20 min at 4 ° C, and dispense the supernatant in a 0.5 ml centrifuge tube. Store at -20 °C.

2.2 protein content detection

After measuring the protein content of each sample using the BCA protein quantification kit (Beijing Kangwei Co., Ltd.), the volume of the solution containing 50 μg of protein was calculated according to the formula, that is, the amount of the sample was loaded.

According to the instructions of the BCA protein test kit, the operation is as follows:

(1) Dilute BSA standard: Dilute BSA standard with RIPA dilution as shown below.

(2) Configuration of BCA working solution: According to the quantity of BSA standard and the sample to be tested, reagent A and reagent B are mixed in a volume ratio of 50:1.

(3) Add 20 μl of the standard to the standard well of a 96-well plate.

(4) Add 2 μl of the sample of the protein to be tested to the well of a 96-well plate, add 18 μl of RIPA lysate, make up to 20 μl of each solution, and mix well, but try not to generate bubbles.

(5) Add 200 μl of the mixture of A and B to the wells of the standard and protein samples, and mix well.

(6) Cover the 96-well plate and incubate at 37 ° C for 30 minutes.

(7) After cooling to room temperature, the absorbance at 562 nm was measured with a microplate reader.

(8) Draw a standard curve and calculate the protein concentration in the sample.

Note: The detection range is: 20-2000μg/ml. If the protein concentration obtained is not checked Within the measurement range, the sample should be diluted again and measured again.

2.3 Electrophoresis

According to the above method, 10% separation gel and 12% concentrated gel are loaded, and the volume is loaded with 50μg protein. The electrophoresis time is generally 2-3 hours, and the voltage is 80V. When the last one of the protein marker can be seen, the electrophoresis can be terminated. Transfer film. After the membrane was transferred, the protein gel was stained with Coomassie Brilliant Blue staining solution for half an hour (on a desalination shaker), and then the staining solution was eluted with a decolorizing solution until the protein bands were clearly observed. Observe whether the target protein band is separated. This method determines the electrophoresis effect.

2.4 transfer film

To transfer a film, prepare 6 filter papers of the same size as the gel and a PVDF film. The order of placement was filter paper-protein gel-PVDF membrane-filter paper. Clamp with a clip and place it in the transfer trough. Note that the protein gel should be contacted with the negative electrode and transferred to a constant flow of 400 mA for 2 hours (the transfer time should be determined according to the size of the target protein band). When the method is used for transfer, heat is generated. Therefore, it is preferable to put an ice box in the transfer tank, and the entire transfer tank is buried in the ice, and the transfer film is passed through.

After the transfer, the film was stained with 1× Lichunhong dye solution for 5 min (on a bleaching shaker). Then, wash the dye solution with double distilled water or TBST solution several times to see the protein on the membrane and observe whether the target protein band is completely transferred. The protein gel can be stained with Coomassie Brilliant Blue for half an hour (on a desalination shaker), and then the dye solution is eluted with a decolorizing solution to see if the protein band on the protein gel is completely transferred. The film transfer effect was determined by the above dyeing method.

2.5 closed

The membrane was soaked from the bottom to the top with TBST, and then transferred to a petri dish containing 5% skim milk powder, and incubated at room temperature for 1 hour with shaking on a shaker.

2.6 Incubating antibodies

The PVDF membrane is blotted with a filter paper, and then placed on a wrap film or a ziplock bag, and the primary antibody and the membrane are sufficiently contacted, and then the wrap film or the ziplock bag is sealed with a sealing machine to prevent the antibody from flowing out. Incubate overnight at 4 ° C shaker. The membrane was placed on a shaker the next day and the PVDF membrane was rinsed 3 times with TBST for 15 min each. The secondary antibody was incubated in the same manner for 1 hour at room temperature. The membrane was placed on a shaker and the PVDF membrane was rinsed 3 times with TBST for 15 min each.

Primary antibody

Secondary antibody

2.7 development

The two reagents A and B were mixed in equal volume on a gel imager, and the membrane protein was brought into full contact with the mixture face down. The PVDF membrane was photographed using Image Lab software of Böhler gel imager, and the gray scale of the target strip was analyzed by ImageJ software.

3. RT-PCR method (Zhou L et al: J Affect Disord. 2013 Sep 25; 150 (3): 776-84; Xiong J et al., Neuro Oncol. 2013 Aug; 15 (8): 990-1007)

3.1 The gene sequences of BDNF, trkB and internal reference β-actin were found from http:www.ncbi.nlm.nih.gov/sites/entrez, and primers were designed using primer premier software.

3.2 Extraction of total RNA: Extraction of total RNA from rat cerebral cortex and hippocampus by Trizol method

1) Extraction method:

Homogenization: After centrifugation, the cells are discarded, the supernatant is discarded, and the cells are lysed by pipetting with 1 ml of Trizol reagent to lyse the cells to a uniform bright liquid state, and the homogenate sample is incubated at 15 to 30 ° C. Minutes to completely decompose the ribosome.

Separation stage: 0.2 ml of chloroform per 1 ml of Trizol. The sample tube cap was capped and the tube was shaken vigorously by hand for 15 seconds and incubated for 2 to 3 minutes at 30 °C. The mixture was centrifuged at 2 to 8 ° C for 15 minutes at a high speed of not more than 12,000 × g. After centrifugation, the mixture was divided into three layers: a lower red phenol-chloroform layer, an intermediate layer, and an upper layer of a colorless aqueous layer. RNA is present in the aqueous layer. The water layer has a capacity of approximately 60% of the added Trizol capacity.

Precipitation of RNA: The aqueous layer was transferred to a clean tube and the RNA was precipitated by mixing the aqueous layer with isopropanol. 0.5 ml of isopropanol per 1 ml of Trizol at the time of initial homogenization. The mixed sample was incubated at 15 to 30 ° C for 10 minutes and centrifuged at 2 to 8 ° C for 10 minutes at a high speed of not more than 12,000 × g. The RNA precipitate is usually invisible before centrifugation, after which a gelatinous pellet is formed which adheres to the tube wall and the bottom of the tube.

Elution of RNA: Remove the supernatant suspension. The RNA was washed once with 75% ethanol and at least 1 ml of 75% ethanol per 1 ml of Trizol. The sample was vortexed and centrifuged at 2 to 8 ° C for 5 minutes at a high speed of not more than 7,500 × g.

Re-dissolution of RNA: At the end of the run, simply dry the RNA pellet. Of particular importance is that it does not allow the RNA to precipitate completely dry, which greatly reduces its solubility. RNA was lysed by pipetting the RNase-free DEPC water several times with a pipette tip and incubating for 10 minutes at 55-60 °C.

2) Total RNA extracted, and its concentration and purity were measured by NanoDrop ND-1000 UV-Vis spectrophotometer. Qualified RNA was stored in a -80 ° C refrigerator.

3.3 RNA reverse transcription to cDNA

The total RNA was less than or equal to 2500 ng using a TAKARA Reverse Transcription Kit (Perfect Real Time) 50 μl system.

RNA reverse transcription temperature system

37 ° C, 15 min

85 ° C, 5 s

4°C, permanent

The cDNA was stored at -20 ° C; while the RNA was stored at -80 ° C.

Based on the measured RNA sample concentration, the amount of RNase-free ddH ₂ O and Total RNA required for the reverse transcription reaction system was calculated. The cDNA product can be used directly as a template for PCR.

4.Golgi staining

A Golgi staining kit from FD NeuroTechnologies, Inc. was used.

(1) Cut the tissue into a 1cm3 square cube (the hippocampus and cortex should be kept intact), immerse it in the mixture of A and B, and change the mixture of A and B after 12 hours at room temperature for 2 weeks. The volume of the soaking solution At least 5 times the size of the organization block.

Note that the A and B mixture should be placed 48 hours in advance, and should be protected from light during the whole process of fixation, dyeing, dehydration and transparency.

(2) Pour off the mixture of A and B, replace it with liquid C, place the refrigerator at 4 °C, change the liquid C after 12 hours, and leave it in the dark for at least 48 hours. The maximum length is no more than one week. Note that both A and B are toxic and cannot be poured directly into the lower pool.

(3) The tissue piece was cut into 150 μm thick pieces by a cryostat. Coronal slice. Take care to avoid light.

(4) Rinse the tissue pieces twice with double distilled water for 2 min each time.

(5) The dyeing solution was prepared in a ratio of D:E liquid: double distilled water at a ratio of 1:1:1. The tissue pieces were then soaked with D, E mixture for 10 min.

(6) Rinse the tissue twice with double distilled water for 4 minutes each time.

(7) Dehydrated with 50%, 75%, and 95% ethanol, respectively, for 4 minutes each time.

(8) Dehydrated 4 times with absolute ethanol for 4 minutes each time.

(9) Transparent three times with xylene for 4 minutes each time.

(10) The plate was sealed with a neutral gum, and observed, and a picture was taken with a confocal microscope.

(11) Calculate the length of dendritic spines using LAS AF Lite software

表示，一组样本经处理前后比较用配对t检验进行检测；两组样本之间的差异用独立样本t检验进行检测，多组资料之间的比较用单因素方差分析检测。P值＜0.05(双尾)被认为有统计学意义。All statistical analyses use version 17.0 SPSS. Experimental data of measurement data with mean ± standard error

A pair of samples were tested before and after treatment by paired t test; the differences between the two groups were tested by independent sample t test, and the comparison between multiple groups of data was detected by one-way analysis of variance. A P value <0.05 (two-tailed) was considered statistically significant.

Seven. Technical effects

A large number of experimental data of the present invention showed that the expression of proBDNF and its receptors p75NTR and Sortilin was up-regulated in the cerebral cortex and hippocampus at the protein level, and the expression of Trkb and BDNF was down-regulated at the protein level. At the molecular transcriptional level, BDNF and TrkB decreased in cortex and hippocampus of depressed rats, while Sortilin and p75 increased.

After intervention in the rat depression model (lateral ventricle injection, intraperitoneal injection, intramuscular injection of antibodies or factors), the behavior of the anti-proBDNF group was significantly improved compared with the NSS group, and the behavior and AVV- of the AVV-BDNF group. The proBDNF and AVV-EGFP groups also showed significant improvement. Through Golgi staining, the formation of depression is positively correlated with the reduction in the length of dendritic spines. The decrease in proBDNF and the increase in BDNF play an important role in the formation of depression and promote the regeneration of neurons in the brain.

The study of this application shows that BDNF content is inseparable from the production of depression. ProBDNF has different biological activities from mature BDNF and is a target for the treatment of affective disorders including depression. Under physiological conditions, hippocampal neurons and some neuroendocrine cells can synthesize and release endogenous pro-BDNF, and pro-BDNF has the opposite effect on mature BDNF. The research results provide a new direction for the treatment of depression, along with the progression to depression A step-by-step study and continuous improvement of the understanding of the functions of neurotrophic factors and their precursor proteins will have important implications for the treatment of depression.

DRAWINGS

In order to more clearly describe the technical solution of the present invention, a brief description will be made below with reference to the accompanying drawings. It is apparent that the drawings are only some of the specific embodiments described herein, and the invention includes, but is not limited to, the drawings. In all the figures, * and # indicate differences, p < 0.05; **, ***, and ### indicate significant differences, p < 0.01.

Figure 1: Normal rats were given different stimulation for 21 days. Depressed rats were injected with anti-proBDNF and NSS (normal goat serum) antibodies in the lateral ventricle, intraperitoneal injection of anti-proBDNF and NSS antibodies, and rat OFT (field test). Quantitative analysis of horizontal motion distances.

Figure 2: Normal rats were given different stimulation for 21 days. Depressed rats were injected with anti-proBDNF and NSS antibodies in the lateral ventricle, intraperitoneal injection of anti-proBDNF and NSS antibodies, and quantitative analysis of the resting time of OFT in rats.

Figure 3: Normal rats were given different stimulation for 21 days. Depressed rats were given anti-proBDNF and NSS antibodies in the lateral ventricle, intraperitoneal injection of anti-proBDNF and NSS antibodies, and quantitative analysis of the number of standing OFT rats.

Figure 4: Normal rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and rat FST (forced swimming test). analysis.

Figure 5: Normal rats were given different stimulation for 21 days. Depressed rats were given intra-cerebral ventricle injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and quantitative analysis of 1% syrup consumption in rats.

Figure 6: Quantitative analysis of FST immobility time in three groups of rats after intramuscular injection of three factors.

Figure 7: Quantitative analysis of OFT immobility time in three groups of rats after intramuscular injection of three factors.

Figure 8: Quantitative analysis of the horizontal distance of OFT in the three groups of rats after intramuscular injection of three factors.

Figure 9: Quantitative analysis of the number of OFT standings in three groups of rats after intramuscular injection of three factors.

Figure 10: Quantitative analysis of 1% syrup water consumption in three groups of rats after intramuscular injection of three factors.

Figure 11a: Changes in expression of proBDNF in the cortex of rats in the depression group and the control group; 11b: The expression of proBDNF in the hippocampus of the depression group and the control group; Figure 11c: Quantitative analysis of the expression of proBDNF in the cortex of the depression group and the control group, *The depression group was different from the control group, p<0.05 Figure 11d: Quantitative analysis of the expression changes of proBDNF in the hippocampus of the depression group and the control group. * The depression group was different from the control group, p<0.05.

Figure 12a: Changes in expression of sortilin in the cortex of rats in the depression group and control group; Figure 12b: Changes in expression of sortilin in the hippocampus region of the depression group and the control group; Figure 12c: Sortilin in the depression group and the control group in the rat cortical region Quantitative analysis of expression changes, * depression group compared with the control group, p <0.05; Figure 12d: quantitative analysis of the expression of sottilin in the hippocampus of the depression group and the control group, * the depression group and the control group were different, p<0.05.

Figure 13a: Changes in expression of p75 in the cortex of rats in the depression group and control group; Figure 13b: Changes in expression of p75 in the hippocampus region of the depression group and the control group; Figure 13c: Depression group and control group p75 in the rat cortical region Quantitative analysis of expression changes, * depression group compared with the control group, p <0.05; Figure 13d: quantitative analysis of the expression of p75 in the hippocampus of the depression group and the control group, * the depression group and the control group were different, p<0.05.

Figure 14a: Changes in expression of Trkb in the rat cortical region of the depression group and control group; Figure 14b: Changes in expression of Trkb in the hippocampus region of the depression group and the control group; Figure 14c: Depression group and control group Trkb in the rat cortical region Quantitative analysis of expression changes, * depression group compared with the control group, p <0.05; Figure 14d: quantitative analysis of the expression of Trkb in the hippocampus of the depression group and the control group, *** depression group compared with the control group Significant difference, p < 0.01.

Figure 15a: Expression of actin in rat cortex and hippocampus; Figure 15b: Expression of BDNF in rat cortex and hippocampus; Figure 15c: Quantitative analysis of BDNF expression in rat cortical areas, **Depression group and control There were significant differences between the groups, p<0.01; Fig. 15d: Quantitative analysis of the expression of BDNF in the hippocampus of rats. *The depression group was different from the control group, p<0.05.

Figure 16a: Expression changes of actin in rat cortex and hippocampus; Figure 16b: Expression changes of TrkB in rat cortex and hippocampus; Figure 16c: Quantitative analysis of expression of TrkB in rat cortical areas, *depression group and control group There were differences, p<0.05; Fig. 16d: Quantitative analysis of TrkB expression in rat hippocampus, *There was a difference between the depression group and the control group, p<0.05.

Figure 17a: Expression of actin in rat cortex and hippocampus; Figure 17b: Sortilin Expression changes in rat cortex and hippocampus; Figure 17c: Quantitative analysis of the expression changes of sortilin in rat cortex, *There was a difference between the depressed group and the control group, p<0.05; Figure 17d: Quantification of the expression of sortilin in the hippocampus of rats Analysis, ** depression group and control group were significantly different, p <0.01.

Figure 18a: Expression changes of actin in rat cortical regions; Figure 18b: Expression changes of actin in rat hippocampus; Figure 18c: Expression of P75 in rat cortical regions; Figure 1Sd: P75 expression in rat hippocampus Changes; Figure 18e: Quantitative analysis of P75 expression changes in rat cortical areas, *Depression group was compared with the control group, p<0.05; Figure 18f: P75 quantitative analysis of expression changes in rat hippocampus, ***Depression group and There was a significant difference in the control group, p < 0.01.

Figure 19: Normal rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and quantitative analysis of dendritic spine length in rat cerebral cortex neurons. .

Figure 20: Normal rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and quantitative analysis of dendritic spine length in rat hippocampal neurons. .

Figure 21a: Golgi staining results of dendritic spine length in cortical neurons of the depressed group; Figure 21b: Golgi staining results of dendritic spine length in the cortical neurons of the control group; Figure 21c: Golgi staining results of dendritic spine length in the hippocampal neurons of the depressed group; Figure 21d : Control group hippocampal neurons dendritic spine length Golgi staining results.

Figure 22a: Golgi staining results of dendritic spine length in rat cortical neurons in the anti-proBDNF group after injection of two antibodies into the lateral ventricle; Figure 22b: Dendritic spine length in rat cortical neurons of the NSS group after injection of two antibodies into the lateral ventricle Golgi staining results; Figure 22c: Golgi staining results of dendritic spine length in hippocampal neurons of anti-proBDNF group after injection of two antibodies into the lateral ventricle; Figure 22d: Hippocampal neuron tree in NSS group after injection of two antibodies into the lateral ventricle Golgi staining results for the length of the spines.

Figure 23a: Golgi staining of dendritic spine length in cerebral cortical neurons of rats in NSS group after intraperitoneal injection of two antibodies; Figure 23b: Dendritic spines of cerebral cortex neurons in anti-proBDNF group after intraperitoneal injection of two antibodies Length Golgi staining results.

Figure 24a: Rat hippocampal neuron dendritic spines in the NSS group after intraperitoneal injection of two antibodies Length of Golgi staining results; Figure 24b: Results of dendritic spine length Golgi staining in hippocampal neurons of rat anti-proBDNF group after intraperitoneal injection of two antibodies.

Figure 25: Quantitative analysis of dendritic spine length in three groups of rat cerebral cortical neurons after intramuscular injection of three factors; ***AVV-proBDNF group and AVV-BDNF group were significantly different, p<0.01;## There was a significant difference between the #AVV-EGFP group and the AVV-BDNF group, p<0.01.

Figure 26a: Golgi staining of dendritic spine length in cerebral cortical neurons of the AVV-BDNF group after intramuscular injection of three factors; Figure 26b: Cerebral cortical neuron tree in the AVV-proBDNF group after intramuscular injection of three factors Results of Golgi staining of the spine length; Figure 26c: Golgi staining results of dendritic spine length in the cerebral cortical neurons of the AVV-EGFP group after intramuscular injection of three factors.

Figure 27: Quantitative analysis of the length of dendritic spines in the hippocampus of three groups of rats after intramuscular injection of three factors; ***AVV-PRPBDNF group and AVV-BDNF group were significantly different, p<0.01;###AVV There was a significant difference between the -EGFP group and the AVV-BDNF group, p<0.01.

Figure 28a: Golgi staining results of dendritic spine length in rat hippocampal neurons of AVV-BDNF group after intramuscular injection of three factors; Figure 28b: Hippocampal neuron tree in rat AVV-proBDNF group after intramuscular injection of three factors Golgi staining results of the length of the spines; Figure 28c: Golgi staining results of dendritic spine lengths in the hippocampal neurons of the AVV-EGFP group after intramuscular injection of three factors.

Figure 29: Effect of intraperitoneal injection of recombinant sortilin ECD-Fc and p75ECD-Fc on depression in rats; ** and *** indicate significant differences in control and dosing of recipient fragments compared to depressed rats, p <0.01.

detailed description

In order to further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with the embodiments. These descriptions are only illustrative of the features and advantages of the invention and are not intended to limit the scope of the invention.

Example 1

Modeling and confirmation of depression rats

A rat model of depression was established by CUMS according to the method described above.

Compared with the control, the results were statistically significant by OFT (including standings, horizontal movement distance and immobility time), FST (immobility time) and SPT (sugar water consumption) behavioral tests (p<0.05 or p<0.01). ), confirming the successful construction of a rat model of depression (Yang CR et al., 2014 Neurotoxicity Research Neurotox Res. 2014 Apr; 25(3): 235-47; Ruan CS et al., Eur J Neurosci. 2014 Aug; 40(4): 2680-90).

Example 2

Protein and transcription level detection

1. Determine actin (SEQ ID NO: 23), proBDNF (SEQ ID NO: 24), sortilin (SEQ ID NO: 26), p75 (actin) in rat cortex and hippocampus according to the above Western blot method. Protein levels of SEQ ID NO: 25) and trkb (SEQ ID NO: 27). The results are shown in Tables 11-17 and Figures 11-14 below.

Western blot results showed that the expression of proBDNF in the hippocampus and cortex of the rats was significantly higher than that of the control group (p<0.05).

Table 11: Comparison of proBDNF expression in the cortex in a rat model of depression

Group proBDNF/actin (%) Control group 0.2754±0.027829 (n=3) Depression group 0.3673±0.01582 ^* (n=6)

* The depression group was different from the control group, p < 0.05.

Table 12: Comparison of proBDNF expression in hippocampus in a rat model of depression

Group proBDNF/actin (%) Control group 0.1239±0.01120 Depression group 0.1886±0.01807 ^*

* The depression group was different from the control group, p < 0.05.

Western blot results showed that the expression of sortilin in the hippocampus and cortex areas of the rats was significantly higher than that of the control group (p<0.05).

Table 13: Comparison of the expression of sortilin in hippocampus in a rat model of depression

Group Sortilin/actin (%) Control group 0.4371±0.1282 (n=3) Depression group 0.8733±0.08611 ^* (n=4)

* The depression group was different from the control group, p < 0.05.

Table 14: Comparison of the expression of sortilin in the cortex in a rat model of depression

Group Sortilin/actin (%) Control group 0.4296±0.06553 (n=3) Depression group 0.6998±0.0477 ^* (n=3)

* The depression group was different from the control group, p < 0.05.

Western blot results showed that the expression of p75 in the hippocampus and cortex areas of the rats was significantly higher than that of the control group (p<0.05).

Table 15: Comparison of p75 expression in cortex in a rat model of depression

Group P75/actin (%) Control group 0.7518±0.04834 (n=3) Depression group 1.434±0.1157 ^* (n=6)

* The depression group was different from the control group, p < 0.05.

Table 16: Comparison of p75 expression in hippocampus in a rat model of depression

Group P75/actin (%) Control group 0.2758±0.04413 (n=3) Depression group 0.4598±0.03708 ^* (n=6)

* The depression group was different from the control group, p < 0.05.

Western blot results showed that the expression of Trkb in the hippocampus and cortex areas of the rats was significantly lower than that of the control group (p<0.05).

Table 17: Comparison of Trkb expression in cortex in a rat model of depression

Group Trkb/actin (%) Control group 0.4751±0.04429 (n=5) Depression group 0.3215±0.03150 ^* (n=6)

* The depression group was different from the control group, p < 0.05.

Table 18: Comparison of Trkb expression in hippocampus in a rat model of depression

Group Trkb/actin (%) Control group 0.5940±0.02050 (n=4) Depression group 0.2037±0.02670 ^*** (n=5)

***There was a significant difference between the depression group and the control group, p<0.01.

2. Determination of actin (SEQ ID NO: 28), BDNF (SEQ ID NO: 29), trkb (SEQ ID NO: 30), sortilin in rat cortex and hippocampus according to the RT-PCR method above. Transcription levels of (SEQ ID NO: 31) and p75 (SEQ ID NO: 32).

The primer sequences of each gene are as follows:

--actin:

Forward primer: 5'-TCACCAACTGGGACG-3' (SEQ ID NO: 21)

Reverse primer: 5'-AGGCATACAGGGACAA-3' (SEQ ID NO: 22)

BDNF:

Forward primer: 5'-GACAAGGCAACTTGGCCTAC-3' (SEQ ID NO: 11)

Reverse primer: 5'-CCTGTCACACACGCTCAGCTC-3' (SEQ ID NO: 12)

TrkB:

Forward primer: 5'-TCATAAGATCCCCCTGGATG-3' (SEQ ID NO: 13)

Reverse primer: 5'-TGCTTCTCAGCTGCCTGAC-3' (SEQ ID NO: 14)

Sortilin:

Forward primer: 5'-ACCAACAATACGCACCAGC-3' (SEQ ID NO: 15)

Reverse primer: 5'-AATAGCCATGCCGAACTCC-3' (SEQ ID NO: 16)

P75:

Forward primer: 5'-AGGGCACATACTCAGACGAA-3' (SEQ ID NO: 17)

Reverse primer: 5'-AGATGGAGCAATAGACAGGAAT-3' (SEQ ID NO: 18)

RT-PCR reaction system, cycle

Actin

2% agarose gel 80v, 5min, 120V, 25min, product: 207 bp.

BDNF

2% agarose gel 80v, 5min, 120V, 25min, product 442bp.

Trk B

2% agarose gel 80v, 5min, 120V, 25min, product: 242 bp.

Sortilin

2% agarose gel 80v, 5min, 120V, 25min, product: 234 bp.

P75

2% agarose gel 80v, 5min, 120V, 25min, product: 324 bp.

RT-PCR gel development, the results are shown in Tables 19-26 and Figures 15-18 below.

In the rat cortex and hippocampus, the transcription level of BDNF in the depression group was significantly lower than that in the control group (p<0.05).

Table 19: Comparison of BDNF/actin (%) in rat cortical regions after 21 days of different stimulation

Group BDNF/actin (%) Control group 1.326±0.01495 (n=3) Depression group 1.138±0.02952 ^** (n=3)

** There was a significant difference between the depression group and the control group, p<0.01.

Table 20: Comparison of BDNF/actin (%) in rat hippocampus after 21 days of different stimulation

Group BDNF/actin (%) Control group 1.233±0.02904 (n=4) Depression group 1.101±0.03376 ^* (n=4)

* The depression group was different from the control group, p < 0.05.

In the rat cortex and hippocampus, the transcription level of TrkB in the depression group was significantly lower than that in the control group (p<0.05).

Table 21: Comparison of TrkB/actin (%) in rat cortical regions after 21 days of different stimulation

Group TrkB/actin (%) Control group 1.076±0.05427 (n=3) Depression group 0.8201±0.07418 ^* (n=3)

* The depression group was different from the control group, p < 0.05.

Table 22: Comparison of TrkB/actin (%) in rat hippocampus after 21 days of different stimulation

Group TrkB/actin (%) Control group 1.233±0.02904 (n=3) Depression group 1.101±0.03376 ^* (n=4)

* The depression group was different from the control group, p < 0.05.

In the rat cortex and hippocampus, the transcription level of sortilin in the depression group was significantly higher than that in the control group (p<0.05).

Table 23: Comparison of stotilin/actin (%) in rat cortical regions after 21 days of different stimulation

Group TrkB/actin (%) Control group 0.6626±0.03712 (n=3) Depression group 0.8437±0.03487 ^* (n=3)

* The depression group was different from the control group, p < 0.05.

Table 24: Comparison of sortilin/actin (%) in rat hippocampus after 21 days of different stimulation

Group Sortilin/actin (%) Control group 0.6489±0.01488 (n=4) Depression group 0.7554±0.01492 ^** (n=4)

** There was a significant difference between the depression group and the control group, p<0.01.

In the rat cortex and hippocampus, the transcription level of P75 in the depression group was significantly higher than that in the control group (p<0.05).

Table 25: Comparison of p75/actin (%) in the rat cortical area after 21 days of different stimulation

Group P75/actin (%) Control group 0.7131±0.02262 Depression group 0.9166±0.06435 ^*

* The depression group was different from the control group, p < 0.05.

Table 26: Comparison of p75/actin (%) in rat hippocampus after 21 days of different stimulation

Group P75/actin (%) Control group 1.255±0.02552 Depression group 1.507±0.02166 ^***

*** There was a significant difference between the depression group and the control group, p<0.01.

Example 3

Intramuscular injection of three factors: AVV8-BDNF, AAV8-proBDNF and AAV8-EGFP

Injection method and dose: 50 μl of AAV8-BDNF, AAV8-proBDNF and AAV8-EGFP were injected into the tibialis anterior muscle of the three groups of depression model mice respectively (Virovek was prepared according to the contract, see Yao et al., Mol Psychiatry., 2015 PMID: 25917367) , 1:1 dilution with PBS, only once. The behavioral test was evaluated two weeks later.

After applying the above three factors, the results of FST, OFT and SPT behavioral experiments were used to detect the results of immobility time, horizontal movement distance and sugar water consumption, respectively, see Table 6-10 and Figure 6-10.

Table 6: Comparison of FST immobility time in three groups of rats after intramuscular injection of three factors

Group No time (s) AVV8-BDNF group 78.71±18.22 AVV8-proBDNF group 180.1±20.33 ^** AVV8-EGFP group 146.3±18.2 ^#

**The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

The #AVV8-EGFP group was different from the AVV8-BDNF group, p<0.05.

Table 7: Comparison of OFT immobility time in three groups of rats after intramuscular injection of three factors

Group No time (s) AVV8-BDNF group 69.44±11.96 AVV8-proBDNF group 137.9±12.69** AVV8-EGFP group 146.0±14.54 ^##

**The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

There was a significant difference between the ##AVV8-EGFP group and the AVV8-BDNF group, p<0.01.

Table 8: Comparison of the horizontal distance of OFT in three groups of rats after intramuscular injection of three factors

Group Horizontal movement distance (m) AVV8-BDNF group 27.73±1.953 AVV8-proBDNF group 11.41±1.193 ^*** AVV8-EGFP group 7.889±1.605 ^###

*** The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

There was a significant difference between the ###AVV8-EGFP group and the AVV8-BDNF group, p<0.01.

Table 9: Comparison of the number of OFT standings in three groups of rats after intramuscular injection of three factors

Group Number of standings (times) AVV8-BDNF group 23.00±2.845 AVV8-proBDNF group 12.71±2.135 ^** AVV8-EGFP group 8.571±1.088 ^###

**The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

There was a significant difference between the ###AVV8-EGFP group and the AVV8-BDNF group, p<0.01.

Table 10: Comparison of 1% syrup consumption in three groups of rats after intramuscular injection of three factors

Group 1% sugar consumption (%) AVV8-BDNF group 80.14±3.203 AVV8-proBDNF group 53.86±6.300 ^* AVV8-EGFP group 50.43±8.443 ^#

* The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.05;

There was a significant difference between the #AVV8-EGFP group and the AVV8-BDNF group, p<0.05.

As described above, after intramuscular injection of three factors, the results of the comparison of Golgi staining are shown below. Table 29-30 and Figure 25-28.

Table 29: Comparison of the length of dendritic spines in the cerebral cortex of three groups of rats after intramuscular injection of three groups of factors

Group Dendritic spine length (μm) AVV8-EGFP group 1.140±0.0749 ^### AVV8-proBDNF group 1.038±0.1073 ^*** AVV8-BDNF group 3.612±0.3224

*** The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

There was a significant difference between the ###AVV8-EGFP group and the AVV8-BDNF group, p<0.01.

Table 30: Comparison of dendritic spine lengths in hippocampal neurons of three groups of rats after intramuscular injection of three groups of factors

Group Dendritic spine length (μm) AVV8-EGFP group 1.096±0.05748 ^### AVV8-proBDNF group 0.7033±0.07482 ^*** AVV8-BDNF group 3.552±0.4913

*** The AVV8-proBDNF group was significantly different from the AVV8-BDNF group, p<0.01;

There was a significant difference between the ###AVV8-EGFP group and the AVV8-BDNF group, p<0.01.

Example 4

1. Preparation of proBDNF and NSS antibodies

Preparation of anti-proBDNF polyclonal antibody

Polyclonal antibodies were prepared using human proBDNF antigens of the following amino acid sequence:

(1) PCR amplification of human proBDNF fragment (reverse primer) 5'-CTAGCGCCGAACCCTCATAGA-3' (SEQ ID NO: 20); forward primer 5'-TTAGCGCCGAACCCTCATAGA-3' (SEQ ID NO: 19), and cloned into the vector pET100/D-TOPO (Invitrogen) Within the site, the plasmid was transfected into E. coli BL21, and the bacterial population was grown in 1000 ml of LB containing 100 μg/ml of ampicillin; shaking was carried out at 300 rpm and 37 ° C, and the OD value of the culture solution at 580 nm was measured. 0.8, then add IPTG to a final concentration of 0.5 mM, incubate at 30 ° C overnight, centrifuge at 1 1000 g at 4 ° C, collect bacteria after 20 minutes; plaque is suspended in 40 ml of buffer, 50 mM potassium phosphate salt Buffer containing 0.3 M sodium chloride, 10% glycerol, 0.005% Triton-X 100, 10 mM imidazole and 1 mM DTT and 1 mM PMSF; Lysozyme was added to the solution to a final concentration of 0.2 mg/ml, and the solution was placed. The cells were lysed on ice for 25 minutes; the solution was then sonicated 10 times for 30 s each time, the power was 50 W, and the whole reaction was placed on ice; again, 11000 g, centrifuged at 4 ° C for 20 minutes; the resulting precipitate was placed in 50 ml. Buffer I (from 20 mM Tris pH 8.0, add 0 .2M sodium chloride and 1% sodium deoxycholate salt); mixed on ice for 30 minutes, the obtained suspension was centrifuged again for 3000 minutes for 10 minutes; the precipitate obtained by centrifugation was placed in 50 ml of ice buffer. II (consisting of 10 mM Tris with pH=8.0, adding 1 mM EDTA and 0.25% sodium deoxycholate), the obtained remission solution was centrifuged again at 3000 g for 10 minutes; the precipitate obtained by centrifugation was washed 3 times with 40 ml of buffer II, after washing Dissolve in 40ml of 8M urea solution; centrifuge the dissolved protein solution at 11000g for 4 minutes at 4°C; add the supernatant to the nickel column, and when all the supernatants are filtered, use 8M urea, 5mM imidazole and 0.5 Wash the nickel column with M sodium chloride washing solution; measure the OD value of the washing liquid until the OD value drops to close to the washing liquid and stop washing; elute with 8M urea, 1M imidazole and 0.5M sodium chloride. The buffer was added to the nickel column to elute the protein of interest. The collected protein was separated by gel electrophoresis and stained with Coomassie blue; the eluate containing the target protein was further neutralized with 0.75 M L-arginine, 5 mM GSH(R). , a solution consisting of 0.5 mM GSSH (O), 5 mM EDTA and 0.1 M Tris (pH = 9.5) to stabilize And white matter and PH; protein solution under conditions 2L PBS at 4 ℃ dialyzed 4 hours, and back to 5L PBS dialyzed for 4 hours and then dialyzed overnight against PBS 10L;

(2) 0.5 mg of the brain-derived neurotrophic factor precursor protein obtained in the step (1) is added to 2 ml of PBS containing 0.4% glutaraldehyde and then 2 ml of Freund's complete adjuvant is added to form an emulsion; it is subcutaneously injected into the back of an adult sheep and Multiple injection sites in the groin; subsequent injections every two weeks, note The dose of the antigen was halved, and incomplete Freund's adjuvant was used until the antibody titer reached 1/10000.

(3) Antibody purification was carried out using a protein G column as described in Fan et al., 2008 Eur J Neurosci. 2008 May; 27(9): 2380-90.

(4) Preparation and purification of normal goat serum immunoglobulin (NSS) for reference See Fan et al., 2008 Eur J Neurosci. 2008 May; 27(9): 2380-90.

2. Administration of antibodies to proBDNF and NSS

2.1 Intraventricular ventricle injection of anti-proBDNF and NSS antibodies (hereinafter referred to as lateral ventricle injection by L)

(1) Establishment of stereotactic surgery model

Rats were intraperitoneally injected with 3% pentobarbital sodium (30 mg/kg), fixed on a brain stereotactic instrument, and the hair at the top of the head was cut off. After disinfection with alcohol, the skin was cut. Refer to Paxinos's rat brain stereo. Positioning map: Select the left lateral ventricle as the injection target area, 1.0mm backward to the midpoint of the anterior humerus, 1.5mm beside the midline, use the dental drill to open the skull, expose the dura mater, and then use the micro syringe to vertically enter the brain surface. The needle was 3.8 mm, at a rate of 10 μl/min (concentration was 1 μg/μl, 20 μg of anti-proBDNF antibody was dissolved in 20 μl of sterilized 0.9% physiological saline), and 20 μg of anti-proBDNF antibody was slowly injected, leaving the needle for 2 min. Slowly withdraw the needle and suture the incision. Strict disinfection to prevent wound infection, while paying attention to the warmth of the rat. The control group was injected with the same amount of NSS antibody.

(2) lateral ventricle positioning

In order to verify the accuracy of the above method, the protein injection (1×) was injected into the lateral ventricle for localization verification before the formal experiment.

After the injection into the lateral ventricle, the horizontal movement distance, the immobility time and the number of standing were detected by the OFT behavioral experiment.

2.2 intraperitoneal injection of anti-proBDNF and NSS antibodies (hereinafter referred to as A by intraperitoneal injection)

Injection method and dose: anti-proBDNF was injected into the peritoneal cavity of the two groups of depression models. And NSS, 1ml/100g (1ml per 100g body weight of rats), 0.08g/ml (0.08g antibody powder per ml of solution), re-injection after 4 days of injection, a total of two injections, the second dose is the first Half of the time. After 20 hours, the OFT behavioral test was performed as above. For comparison results, see Table 1-3 and Figures 1-3.

Table 1: Different rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and the horizontal distance of rat OFT was compared.

Group Horizontal movement distance (m) Control group 19.68±0.9478 (n=11) Depression group 11.33±1.144 (n=11) L-anti-proBDNF group 22.26±2.316 (N=10) L-NSS group 8.779±2.178 (N=10) A-anti-proBDNF group 16.58±0.9711 (N=6) A-NSS group 6.729±1.769 (N=6)

***The control group was different from the depression group, p<0.01; the *** control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

*** The L-anti-proBDNF group was different from the depression group (p<0.01);

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

*** The L-anti-proBDNF group was different from the A-NSS group, p<0.01;

*A-anti-proBDNF group was different from depression group, p<0.05;

**A-anti-proBDNF group was different from L-NSS group, p<0.01; and

**A-anti-proBDNF group was different from A-NSS group, p<0.01.

Table 2: Different rats were given different stimulation for 21 days. The rats in the depression were given anti-proBDNF and NSS antibodies in the lateral ventricle, and anti-proBDNF and NSS antibodies were injected intraperitoneally.

Group No time (s) Control group 70.88±3.848 (N=11) Depression group 206.4±22.46 (N=11) L-anti-proBDNF group 66.44±16.88 (N=10) L-NSS group 208.4±20.74 (N=10) A-anti-proBDNF group 117.7±12.01 (N=6) A-NSS group 209.5±16.40 (N=6)

***The control group was different from the depression group, p<0.01;

***The control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

*** L-anti-proBDNF group and depression group were different, p <0.01;

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

*** The L-anti-proBDNF group was different from the A-NSS group, p<0.01;

***A-anti-proBDNF group was different from depression group, p<0.01;

**A-anti-proBDNF group was different from L-NSS group, p<0.01; and

**A-anti-proBDNF group was different from A-NSS group, p<0.01.

Table 3: Normal rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and the number of standing OFT rats was compared.

Group Number of standings (times) Control group 22.33±1.900 (N=11) Depression group 4.222±1.103 (N=11) L-anti-proBDNF group 18.40±1.568 (N=10) L-NSS group 5.80±1.020 (N=10) A-anti-proBDNF group 18.00±0.9309 (N=6) A-NSS group 5.400±1.208 (N=6)

***The control group was different from the depression group, p<0.01;

***The control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

*** L-anti-proBDNF group and depression group were different, p <0.01;

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

*** The L-anti-proBDNF group was different from the A-NSS group, p<0.01;

***A-anti-proBDNF group was different from depression group, p<0.01;

***A-anti-proBDNF group was different from L-NSS group, p<0.01;

*** The A-anti-proBDNF group was different from the A-NSS group, p<0.01.

As described above, after injection of antibodies into the lateral ventricle and the peritoneal cavity, the immobility time and the syrup consumption were measured by FST and SPT behavioral experiments, respectively. For the comparison results, see Table 4-5 and Figure 4-5.

Table 4: Different rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and the FST immobility time of rats was compared.

Group Less time (s) Control group 86.64±9.059 (N=11) Depression group 200.5±17.10 (N=11) L-anti-proBDNF group 101.8±13.79 (N=10) L-NSS group 173.6±3.516 (N=10) A-anti-proBDNF group 90.20±25.20 (N=6) A-NSS group 194±29.58 (N=6)

***The control group was different from the depression group, p<0.01;

**The control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

**The L-anti-proBDNF group was different from the depression group, p<0.01;

*L-anti-proBDNF group was different from L-NSS group, p<0.05;

**The L-anti-proBDNF group was different from the A-NSS group, p<0.01;

***A-anti-proBDNF group was different from depression group, p<0.01;

*A-anti-proBDNF group was different from L-NSS group, p<0.05; and

**A-anti-proBDNF group was different from A-NSS group, p<0.01.

Table 5: Different rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and 1% sucrose consumption in rats.

Group 1% sugar consumption (%) Control group 0.8918±0.05001 (N=11) Depression group 0.3653±0.07435 (N=11) L-anti-proBDNF group 0.7240±0.01435 (N=10) L-NSS group 0.1540±0.06794 (N=10) A-anti-proBDNF group 0.9317±0.04968 (N=6) A-NSS group 0.3740±0.1716 (N=6)

***The control group was different from the depression group, p<0.01;

***The control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

**The L-anti-proBDNF group was different from the depression group, p<0.01;

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

*L-anti-proBDNF group was different from A-NSS group, p<0.05;

***A-anti-proBDNF group was different from depression group, p<0.01;

***A-anti-proBDNF group was different from L-NSS group, p<0.01; and

*** The A-anti-proBDNF group was different from the A-NSS group, p<0.01.

As described above, the antibody was injected into the lateral ventricle and the peritoneal cavity, and the results of the Golgi staining before and after administration are shown in Tables 27-28 and 19-24 below.

Table 27: Different rats were given different stimulation for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and the length of dendritic spines in rat cortical regions.

Group Dendritic spine length (μm) Control group 2.770±0.2553 (N=11) Depression group 0.7583±0.2485 (N=11) L-anti-proBDNF group 2.571±0.2832 (N=10) L-NSS group 1.137±0.07868 (N=10) A-anti-proBDNF group 2.475±0.1794 (N=6) A-NSS group 1.392±0.1390 (N=6)

***The control group was different from the depression group, p<0.01;

***The control group was different from the L-NSS group, p<0.01;

**The control group was different from the A-NSS group, p<0.01;

*** The L-anti-proBDNF group was different from the depression group, <0.01;

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

**The L-anti-proBDNF group was different from the A-NSS group, p<0.05;

***A-anti-proBDNF group was different from depression group, p<0.01;

***A-anti-proBDNF group was different from L-NSS group, p<0.01; and

**A-anti-proBDNF group was different from A-NSS group, p<0.01.

Table 28: Different rats were given different stimulations for 21 days. Depressed rats were given intracerebroventricular injection of anti-proBDNF and NSS antibodies, intraperitoneal injection of anti-proBDNF and NSS antibodies, and the length of dendritic spines in rat hippocampus.

Group Dendritic spine length (μm) Control group 2.892±0.2017 (N=11) Depression group 0.7317±0.2390 (N=11) L-anti-proBDNF group 2.716±0.2070 (N=10) L-NSS group 1.136±0.09440 (N=10) A-anti-proBDNF group 2.495±0.07343 (N=6) A-NSS group 1.266±0.08959 (N=6)

***The control group was different from the depression group, p<0.01;

***The control group was different from the L-NSS group, p<0.01;

***The control group was different from the A-NSS group, p<0.01;

*** L-anti-proBDNF group and depression group were different, p <0.01;

*** L-anti-proBDNF group and L-NSS group were different, p <0.01;

*** The L-anti-proBDNF group was different from the A-NSS group, p<0.05;

***A-anti-proBDNF group was different from depression group, p<0.01;

***A-anti-proBDNF group was different from L-NSS group, p<0.01; and

*** The A-anti-proBDNF group was different from the A-NSS group, p<0.01.

Example 5

Effects of recombinant sortilin ECD-Fc (SEQ ID NO: 8) and p75ECD-Fc (SEQ ID NO: 9) on depression in rats

SD male rats, 2-3 months old, were divided into four groups, n=10:1. normal control group; 2. depression group + standard saline; 3. depression group + sortilin ECD-Fc; Depressive group + p75ECD-Fc.

Rats were subjected to CUMS for three weeks in all depression groups. Two weeks after CUMS, on day 14, rats were injected intraperitoneally with standard saline at a dose of 10 mg/kg, or with recombinant sortilin ECD-Fc or p75ECD-Fc (prepared by cloning with human immunoglobulin fragment C). The fused p75 extracellular domain or the sortilin extracellular domain fragment (SEQ ID NO: 7), CHO cells were expressed and purified via a protein G column), and on day 18, the rats were repeatedly injected with the above agent, but the dose was halved. 5mg/kg. On day 22, all rats were tested for sugar water consumption and forced swimming. The sugar concentration is calculated based on the total amount of water and sugar water consumed. The results (see Figure 29) show that injection of sortilin ECD-Fc or p75ECD-Fc blocked the reduction in sugar water consumption and the increase in immobility time in forced swimming experiments.

All of the documents mentioned above are incorporated herein by reference in their entirety as if they were individually incorporated by reference. The above description of the embodiments is merely for helping to understand the core idea of the present invention. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention, but these modifications and modifications are also claimed in the appended claims. In the range.

Claims (19)

The use of a binding molecule having neutralizing or inhibitory activity against a brain-derived neurotrophic factor precursor protein (proBDNF) or a signaling molecule thereof for the preparation of a medicament for preventing, alleviating or treating a affective disorder. The use of claim 1, wherein the signaling molecule is the receptor p75NTR, sortilin or a fragment thereof of proBDNF. The use of claim 1 or 2, wherein the affective disorder is selected from the group consisting of depression and anxiety. The use of claim 1, wherein the binding molecule is selected from a binding molecule that inhibits and/or blocks expression of a gene of proBDNF or a signaling molecule thereof, specifically binds to a binding molecule of proBDNF or a signaling molecule thereof, or promotes proBDNF cleavage. Binding molecule of BDNF. The use of claim 4, wherein the binding molecule that inhibits and/or blocks gene expression is selected from the group consisting of an antisense nucleic acid, siRNA, shRNA, dsRNA, miRNA or ribozyme. The use of claim 4, wherein the binding molecule that facilitates cleavage of proBDNF into mature BDNF is selected from the group consisting of furin, matrix metalloproteinase (MMP) 2/7/9, and tissue plasminogen activator (tPA). The use of claim 4, wherein the binding molecule that specifically binds to proBDNF or a signaling molecule thereof is selected from the group consisting of an antibody, a receptor p75 that binds to proBDNF, orortilin or a fragment thereof, and functional variants thereof. The use of claim 2 or 7, wherein the acceptor fragment is selected from the group consisting of p75ECD-Fc and sortilin ECD-Fc. The use of claim 7, wherein the functional variant is selected from the group consisting of chemically modified variants, substituted, added or deleted variants. The use of claim 7, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a murine antibody or a fragment thereof. The use according to claim 10, wherein the fragment is selected from the group consisting of Fab, F(ab'), F(ab') ₂ , Fv, dAb, Fd, complementarity determining region (CDR) fragment, single chain antibody (scFv), A bivalent single chain antibody, a single chain phage antibody, a bispecific diaborating antibody, a triple chain antibody or a four chain antibody. The use of claim 10, wherein the polyclonal antibody is produced by immunizing an animal with proBDNF or a fragment thereof. The use of claim 12, wherein the fragment is the amino acid sequence set forth in SEQ ID NO: 10. The use of claim 1 or 2, wherein the binding molecule is an antagonist that prevents proBDNF from binding to its receptor sortilin or p75. The use of a binding molecule that stimulates or promotes BDNF or its signaling molecule by restoring BDNF/proBDNF signaling balance in the preparation of a medicament for the prevention, alleviation or treatment of affective disorders. The use of claim 15, wherein the binding molecule is BDNF or its signaling molecule itself. The use of claim 15, wherein the binding molecule is the receptor TrkB of BDNF. The use of claim 15 wherein said binding molecule is BDNF or its receptor TrkB The sense nucleic acid. The use of claim 15, wherein the binding molecule is an agonist of BDNF or TrkB.

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