WO2011140997A1 - Transgenic drosophila model of alzheimer's disease and use thereof in drug screening - Google Patents

Abstract

The present invention belongs to the technical field of transgenic animal model and discloses a transgenic drosophila model, for screening therapeutic drugs for Alzheimer's disease, the construction method and the use thereof. The purpose of the present invention is to provide a transgenic drosophila model, for investigating the molecular pathways of the development process of Alzheimer's disease and screening drugs. In the present invention, stable and heritable drosophila strains are obtained by successive hybridization of the existing transgenic drosophila. The said model can simulate the in vivo generating and metabolic process of the major pathogenic genes of Alzheimer's disease.

Description

 Alzheimer's disease transgenic fruit fly model and its application in drug screening

Technical field

 The invention belongs to the field of medicine and biology, in particular, the invention relates to research on the shearing action of the pathogenic gene APP of Alzheimer's disease (AD) in vivo and the product β-amyloid egg in the pathogenesis Molecular mechanisms and a novel Drosophila model for screening new drugs for the treatment of AD. technical background

 Alzheimer's disease (AD) is a central nervous system degenerative disease. In recent years, it has become the fourth biggest killer after vascular disease, cancer and stroke. According to the World Health Organization, the number of Alzheimer's patients worldwide is estimated to be around 18 million. Patients with Alzheimer's disease are clinically characterized by generalized dementia such as memory impairment, aphasia, agnosia, loss of recognition, visual spatial impairment, executive dysfunction, and personality and behavioral changes.

 The histopathological manifestations of AD are mainly senile plaques (SP), neurofibrillary tangles (NFTs), and regional neuronal cell death caused by apoptosis. The main component of amyloid plaques is β-amyloid (Αβ). The pathogenesis of Alzheimer's disease is not well understood. There are currently three hypotheses: the beta amyloid cascade hypothesis, the Tau protein hypothesis, and the angiogenic hypothesis. Among them, the β amyloid cascade hypothesis is dominant, and the research is relatively mature.

--amyloid protein is produced by sequential cleavage of amyloid precursor protein (APP) by β-secretase (BACE) and γ-secretase (γ-secretase). Intracerebral Αβ is derived from its precursor substance, amyloid precursor protein (APP). The process of metabolizing from APP to amyloid beta peptide is divided into two steps: First, β-secretase cleaves sputum at the Ν end of Αβ, producing a soluble secreted quinone derivative APPS-β and a C-terminal fragment of the transmembrane component. The C-terminal fragment is further cleaved by γ-secretase to Aβ. Under certain pathological conditions, sputum is mainly cleaved by β-secretase and γ-secretase to produce excessive Αβ, leading to AD disease. Γ-secretase plays a very important role in the production of Aβ, which determines the proportion of Aβ42 produced therein. Now think that this enzyme is It consists of four transmembrane proteins consisting of presenilin, nicastrin, APH1 and Pen2. Presenilin is a premature protein, a catalytic subunit of γ-secretase. It has been found that more than one hundred mutations in the gene encoding presenilin 1 can cause familial senile dementia. The pathogenesis is probably due to mutant changes. Γ-secretase activity increases the proportion of Αβ42 production.

 In the past few decades, people have successfully modeled a variety of neurodegenerative diseases in animals through transgenic mouse models, which has led to a deep understanding of these diseases and treatment. However, most of the transgenic mouse models used in the study of AD require a lot of time and money, which seriously affects the progress of the research.

 Since the discovery of melanoma cell tumors using the Drosophila model in 1918, humans have made significant breakthroughs in the study of tumors, neurodegenerative diseases, sleep disorders, diabetes, and obesity using the Drosophila model. The new Drosophila model has been used to study Hereditary spastic paraplegia (HSP), Spinocerebellar ataxia (SCA), Leukemia, PDGF/VEGF receptor molecule (PVR) genes, etc. More human genetic and degenerative diseases can be replicated using fruit flies.

 The transgenic Drosophila model has many unique advantages in the study of degenerative diseases represented by AD. First, in the known pathogenic genes of 714 genetic diseases in humans, Drosophila has 548 homologous genes, such as Amyloid percursor protein like (APPL) and Presenilin (AD), huntingtin (Huntington's disease, HD, Huntington). Disease) and so on. In addition to a large number of homologous genes, Drosophila's neurodegenerative disease model has many similar phenotypes as human neurodegenerative diseases, such as late onest, progressive, and high nervous system. toxicity. Drosophila has an amyloide precursor protein like (APPL), which is homologous to human APP. Luo experiments confirmed the defect in behavioral phenotype of the fruit fly that knocked out the APPL gene. It can be improved after the transfer of the APPL gene. The expression of the APPL gene mutation can compensate for the lack of behavioral testing in rats, and changes in synaptic variability, axonal transport, and apoptosis after APP gene mutation can also be observed. Due to the short life cycle of Drosophila, the inventors were able to study the whole process of neurodegenerative diseases in the Drosophila model in a short time.

It can be seen that the existing Αβ Drosophila model and the high similarity of Drosophila endogenous PS 1 protein to humans are used to reconstruct the production process of Αβ based on prion-based cleavage in Drosophila. These established AD fruit fly models reproduce the main pathological features of AD disorders, including shortened life spans. Defects in dynamism and defects in learning and memory, as well as neurotoxicity due to the expression of Αβ. Crowther et al. demonstrated that MK-801, an antagonist of glutamate receptors, can extend the lifespan of AD fruit flies. Iijima et al. demonstrated that Congo red, a substance that blocks Αβ deposition, can significantly alleviate the behavioral deficits of AD fruit flies and neuronal damage caused by Αβ toxicity. Therefore, the present inventors can study the interaction of Αβ and ΑΡΡ and related secretases in the process of AD in Drosophila, and conduct drug screening studies targeting various proteins involved in the process, thereby revealing pathogenic related genes. Possible links with Alzheimer's disease.

 Therefore, it is necessary to develop a new suitable fruit fly model in the field, which will help to provide a new way for the research and treatment of AD. Summary of the invention

 The present invention provides a method for preparing a Drosophila model, characterized in that: the method comprises: (1) hybridizing a UAS-APP strain of Drosophila with a DB strain of Drosophila, and subfamily Drosophila backcrossing with DB to obtain 2 The chromosome is APP/Cyo, and the chromosome 3 is ΤΜ6Β/ΤΜΠ;

 (2) The UAS-BACE strain Drosophila is crossed with the DB strain Drosophila, and the Drosophila fruit flies are backcrossed with DB to obtain Drosophila with chromosome 2 as Bl/Cyo and chromosome 3 as BACE/TM6B;

 (3) Crossing the Drosophila lines obtained in steps (1) and (2), and then self-crossing the resulting Drosophila chromosome 2 as APP/Cyo and chromosome 3 as BACE/TM6B to obtain chromosome 2 It is a homozygous APP/APP, and the chromosome 3 is a homozygous BACE/BACE stable genetic flies;

 (4) The UAS-BACE; DPsn strain Drosophila was crossed with the DB strain of Drosophila, and then backcrossed with the DB strain to obtain Drosophila with chromosome 2 as Bl/Cyo and chromosome 3 as DPsn/TM6B;

 (5) The Drosophila obtained in the step (1) is crossed with the Drosophila obtained in the step (4) to obtain a fruit fly with the chromosome 2 as APP/Cyo and the chromosome 3 as DPsn/TM6B;

 (6) The Drosophila obtained in the step (5) is hybridized with the chromosome 2 obtained in the step (3) as the APP/Cyo, and the chromosome 3 is the BACE/TM6B progeny, and the chromosome 2 is APP/Cyo. Chromosome recombination of chromosome 3, DPsn-BACE/DPsn-BACE, which exhibits three traits of Drosophila;

 (7) The Drosophila obtained in the step (6) is crossed with DB to obtain a fruit fly with chromosome 2 as APP/Cyo and chromosome 3 as DPsn-BACE/TM6B;

(8) Self-crossing the fruit fly obtained in step (7), obtaining chromosome 2 as homozygous APP/APP, No. 3 The chromosome is a homozygous DPsn-BACE/DPsn-BACE stable genetic flies, resulting in a Drosophila model.

 In an embodiment, the method further includes:

 (9) The Drosophila obtained in the step (8) is crossed with the DB, and the progeny Drosophila is crossed back with +/+; TM6B/TMII, and the chromosome 2 of the obtained fruit fly is +/+ , chromosome 3 is

DPsn-BACE/DPsn-BACE; The Drosophila expresses both β-secretase and presenilin but does not express the substrate protein ΑΡΡ, thereby producing a double-traited fruit fly.

 In an embodiment, the method further includes:

 (10) hybridizing the Drosophila obtained in the step (8) with GAL4 (cha) to obtain a progeny capable of specifically expressing the target protein in the cholinergic neuron; or

(11) The Drosophila obtained in the step (8) is hybridized with GAL4 (el _av ) to obtain a progeny which can specifically express the target protein in the whole neuron.

 The present application provides a Drosophila somatic cell or tissue that co-expresses APP protein and beta secretase (BACE) and does not overexpress or overexpress Drosophila presenilin protein (Dpsn).

 In one embodiment, the Drosophila somatic cell or tissue is obtained by employing the step (10) or

(11) The method of making fruit flies.

 In one embodiment, the tissue is selected from the group consisting of neural tissue of a fruit fly.

 In one embodiment, the tissue is selected from brain tissue of a fruit fly.

 The present application provides the use of Drosophila, its somatic cells or tissues as described herein for studying the interaction of the substrate APP and its cleavage β-secretase and γ-secretase, the intermediate cleavage product βΑΡΡ-CTF (C99) And the role of the final product in the pathological process of the disease associated with the overexpression of β-silver protein, or the correlation between the prion cleavage process and the β-amyloid overexpression-related disease, or the study of ΑΡΡ protein The process of shearing and the overexpression of β-amyloid in pathological processes in specific neuronal tissues.

 The present application provides the use of Drosophila, its somatic cells or tissues as described herein for screening β-secretase, γ-secretase and/or β-amyloid in the process of β-amyloid overexpression-related diseases The drug to be used; or a drug for screening for a disease associated with β-amyloid overexpression by regulating the expression of β-secretase and/or γ-secretase and a target protein interacting therewith.

The present application also provides a Drosophila, a somatic cell or tissue thereof as described herein for use in the preparation of a beta-amyloid. Use of a drug for overexpressing a protein associated with a disease.

 In one embodiment, the β-amyloid overexpression related disorder is a neurodegenerative disease. In one embodiment, the neurodegenerative disease is Alzheimer's disease.

 The present application provides a method of screening for potential substances that ameliorate diseases associated with β-amyloid overexpression, including:

 (1) contacting the candidate substance with a system expressing a β-secretase protein or a γ-secretase component protein;

(2) detecting the effect of the candidate substance on the β-secretase protein or the γ-secretase component protein;

 Wherein, if the candidate substance can reduce the expression or activity of the β-secretase protein or the γ-secretase component protein, it indicates that the candidate substance is a potential substance which can be used to improve the disease associated with β-amyloid overexpression.

 In a preferred embodiment, in the above step (2), if the candidate substance can reduce the expression or activity of the β-secretase protein or the γ-secretase component protein, and does not completely inhibit the β-secretase protein or γ- Expression of the secreted enzyme component protein indicates that the candidate substance is a potential substance useful for ameliorating diseases associated with β-amyloid overexpression.

 In another preferred embodiment, the method further comprises: expressing a target protein in a specific brain tissue region, wherein the candidate substance can alleviate the toxicity of β-amyloid overexpression, indicating that the candidate substance is useful for improving β- Amyloid overexpression of potential substances associated with diseases.

 In another preferred embodiment, the method comprises: adding a candidate substance to a system expressing a β-secretase protein or a γ-secretase component protein in a test group; and/or

 The step (2) comprises: detecting the expression or activity of the β-secretase protein or the γ-secretase component protein in the system of the test group, and comparing with the control group, wherein the control group is not added with the candidate substance a system for expressing a β-secretase protein or a γ-secretase component protein;

 If the expression and activity of the β-secretase protein or the γ-secretase component protein in the test group are statistically lower (; preferably significantly lower than, for example, 10% or more lower, preferably 20% or more lower; more preferably; The lower than 40% of the control group indicates that the candidate substance is a potential substance that can be used to improve the disease associated with β-amyloid overexpression.

 In another preferred embodiment, the system is selected from the group consisting of: a cell system (or a cell culture system;), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

In another preferred embodiment, the method further comprises: performing further cells on the obtained potential substance Experimental and/or animal experiments to further select and determine from the candidate substances substances useful for ameliorating diseases associated with β-amyloid overexpression.

 The present application also provides a method of screening for potential substances that ameliorate a disease associated with β-amyloid overexpression, the method comprising:

 The Drosophila model was prepared according to the method for preparing a Drosophila model as described herein.

 Giving the Drosophila model candidate substance; and

 Detecting the effect of the candidate substance on the β-secretase protein or the γ-secretase component protein in the Drosophila model, or detecting the behavior of the Drosophila model;

 Wherein, if the candidate substance can reduce the expression or activity of the β-secretase protein or the γ-secretase component protein in the Drosophila model, it indicates that the candidate substance is useful for improving the β-amyloid overexpression related diseases. Potential substance; or, if the candidate substance alters the behavior, survival rate, and/or memory capacity of the Drosophila model, indicating that the candidate substance is useful for improving neurodegenerative diseases caused by β-amyloid overexpression The underlying substance of the symptoms.

 In one embodiment, the Drosophila model co-expresses prion protein and beta secretase (BACE) and does not express or overexpress Drosophila presenilin protein (Dpsn).

 The present application also includes a Drosophila model obtained by the preparation method of the present application, a somatic cell or tissue thereof, and a substance screened by the screening method of the present application and a preparation thereof for treating a β-amyloid overexpression-related disease (for example, neurodegenerative Use in drugs for sexually transmitted diseases such as Alzheimer's disease. As used herein, when used to define pharmaceutical use, "preparation" includes not only the preparation of the active ingredient into a drug, but also the process of development, pharmaceuticals, etc. of the drug prior to shipment.

 Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

Figure 1 shows the specific process for establishing the Drosophila research model of the present invention. Α, the UAS-APP strain Drosophila is crossed with the DB strain Drosophila, and the Drosophila fruit fly back to DB to obtain the Drosophila process of chromosome 2 as APP/Cyo and chromosome 3 as ΤΜ6Β/ΤΜΠ. B. The UAS-BACE strain Drosophila is crossed with the DB strain Drosophila, and the Drosophila fruit fly back to DB to obtain the Drosophila process of chromosome 2 as Bl/Cyo and chromosome 3 as BACE/TM6B. C. After the Drosophila lines obtained in Figure A and Figure B are crossed, the offspring fruit flies are self-fertilized. The chromosome is homozygous APP/APP, and chromosome 3 is a process of homozygous BACE/BACE stable genetic inducing Drosophila model. D. Isolation of UAS-DPsn fruit flies, hybridization of UAS-BACE/DPsn strains of Drosophila with DB strains of Drosophila, and backcrossing with DB, obtaining chromosome 2 as Bl/Cyo and chromosome 3 as DPsn/TM6B The process of the fly. E. The process of obtaining a fruit fly with chromosome 2 as APP/Cyo and chromosome 3 as DPsn/TM6B. F. The process of obtaining a Drosophila strain in which chromosome 2 is APP/Cyo and chromosome 3 recombines DPsn-BACE/TM6B. G. The process of obtaining a homozygous APP/APP for chromosome 2 and a stable genetic Drosophila model for homozygous DPsn-BACE/DPsn-BACE.

 Figure 2 shows the expression of human APP protein, β-secretase protein in Drosophila, and the identification of Drosophila model. A. Schematic diagram of the cleavage site of β-secretase and γ-secretase on APP protein. Β, human ΑΡΡ, β-secretase protein is expressed in Drosophila and can be cleaved to produce βΑΡΡ-C-terminal products. C, AD transgenic Drosophila β-amyloid protein production by enzyme-linked immunosorbent assay quantitative detection results. D. The results of β-amyloid precipitation and immunofluorescence can be formed in Drosophila.

 Figure 3 shows the apparent defects in the behavior of AD Drosophila, and the reduction of β-secretase protein activity can alleviate the symptoms of AD Drosophila to a certain extent. Α, Κ-Μ curve to observe the survival index of male Drosophila of various strains, (a) is the expression of cholinergic neurons, (; b) is the expression of whole neurons; B, obtained according to the results in Figure A A bar chart of the lifespan of each fruit fly; C. Crawling test to test the mobility of various fruit fly strains; among them, the ability index indicates the crawling rate of each parallel experimental fruit fly; 0 and 5, reduce β-secretion Enzyme protein activity can improve the viability of AD fruit flies to a certain extent.

Figure 4 shows that Aricept and sputum can significantly alleviate the behavioral deficits of AD fruit flies. Α and Arison can significantly prolong the lifespan of AD fruit flies and alleviate the defects in the ability of AD Drosophila to act (the model of cholinergic neuron expression). Among them, Aricept can significantly prolong Αβ compared with the solvent group Drosophila. The lifespan of transgenic AD fruit flies. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ安理申 can significantly prolong the lifespan of Αβ transgenic fruit flies, but the effect of ΑΡΡ/secretase transgenic AD fruit flies is not obvious. Compared with the solvent group Drosophila, Aricept can significantly alleviate the deficiencies in the ability of Αβ transgenic AD Drosophila and ΑΡΡ/secretase transgenic AD Drosophila. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ 安理申 can significantly promote the activity of Αβ transgenic Drosophila and ΑΡΡ/secretase transgenic AD Drosophila, 30μΜ 安理申 effect is more significant. Β and Arison can significantly prolong the lifespan of AD fruit flies and alleviate the defects in AD Drosophila's ability to act (a whole neuron expression model). Among them, Aricept can significantly prolong Αβ transgenic AD compared with the solvent group Drosophila. The lifespan of fruit flies. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ安理申 can significantly prolong Αβ turn The lifespan of the gene Drosophila, but the effect of the APP/secretase transgenic AD fruit fly is not obvious. Compared with the solvent group Drosophila, Aricept can significantly alleviate the deficiencies in the ability of Αβ transgenic AD Drosophila and APP/secretase transgenic AD Drosophila. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ 安理申 can significantly promote the activity of Αβ transgenic Drosophila and ΑΡΡ/secretase transgenic AD Drosophila, 30μΜ 安理申 effect is more significant. C and NTI can significantly prolong the lifespan of AD fruit flies and alleviate the defects in the action ability of AD Drosophila (the expression model of cholinergic neurons;), NTI can significantly prolong the Αβ transgene compared with the solvent group Drosophila. The lifespan of AD Drosophila and APP/secretase transgenic AD flies. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ of NTI can significantly promote the longevity of Αβ transgenic Drosophila and APP/secretase transgenic AD flies. Compared with the solvent group Drosophila, NTI can significantly alleviate the defect in the ability of Αβ transgenic AD Drosophila and APP/secretase transgenic AD Drosophila. η = 6, *Ρ < 0.05. ΙΟμΜ and 30μΜ of NTI can significantly promote the activity of Αβ transgenic Drosophila and APP/secretase transgenic AD Drosophila, and the 30μΜ NTI effect is more significant. D, NTI can significantly prolong the lifespan of AD Drosophila, and alleviate the defects in AD Drosophila's ability to move (a whole neuron expression model). Among them, NTI can significantly prolong the Αβ transgenic AD fruit fly compared with the solvent group Drosophila. And APP/secretase transgenic AD fruit fly life. η = 6, *Ρ < 0.05. Both ΙΟμΜ and 30μΜ of NTI can significantly promote the longevity of Αβ transgenic Drosophila and APP/secretase transgenic AD flies. Compared with the solvent group Drosophila, NTI can significantly alleviate the defect in the ability of Αβ transgenic AD Drosophila and ΑΡΡ/secretase transgenic AD Drosophila. η = 6, *Ρ < 0.05. ΙΟμΜ and 30μΜ NTI can significantly promote the activity of Αβ transgenic Drosophila and APP/secretase transgenic AD Drosophila, and the 30μΜ NTI effect is more significant. Ε, Arijan and NTI can improve the learning and memory ability of AD Drosophila to a certain extent.

 Figure 5 shows that Aricept and NTI do not affect the expression of exogenous Αβ, which can alleviate the plaque formation of neuronal damage caused by β-amyloid precipitation. Α, immunoblotting to detect the β-amyloid deposition in the brain of the fly by the Anritsu and NTI treatment; Β, immunofluorescence to observe the expression of Αβ in the Drosophila brain of each strain after treatment with Arison and NTI; C, Sumu The sperm-eosin staining was used to detect the plaque of neuronal damage in the brains of Drosophila in each strain after treatment with Arison and NTI. detailed description

After intensive research, the inventors have for the first time developed a new animal model using Drosophila as a research object, which expresses human APP protein and β-secretase protein in Drosophila Reproducing the production process and pathological features of β-amyloid in vivo provides a new way for the study of neurodegenerative diseases. Drosophila animal model

 In order to study the role of β-amyloid in the occurrence or development of neurodegenerative diseases, the inventors have conducted extensive research to determine that the complete system of β-amyloid precursor peptone and its cleavage enzyme formation is a very Important influencing factors. On the basis of this, the inventors further developed an animal model suitable for studying the β-amyloid production process.

 In order to obtain a suitable animal model, the present inventors have conducted extensive research work on the selection of animal species, and finally determined to prepare an animal model using Drosophila. First, the transgenic flies are used to study neurodegenerative diseases, and the pathological symptoms are strikingly similar to those exhibited in patients. Secondly, the genetic background of Drosophila is clear, the genetic manipulation is simple, the life cycle is short, and consumption Less, these characteristics make Drosophila a convenient tool for studying neurodegenerative diseases; third, human Psn protein (see GenBank accession number: NM_000021.3) and Drosophila DPsn protein (see GenBank accession number: NM 079460.2) The homology is high and the identity is over 50%. The human APP protein (see GenBank accession number: NM_201414. 1) is also highly homologous to the Drosophila APPL protein (see GenBank accession number: NM-057278.3), with 40% identity, indicating their conservation. It can be seen that the use of fruit fly research can accurately reflect the actual situation of human beings and has good reference significance. These characteristics make Drosophila a useful tool for studying the regulation of β-amyloid production and for screening new neurodegenerative diseases (such as AD).

After determining the animal model, the inventors further determined a model for establishing a model for the production of sputum and splicing enzymes, in vivo reproducible enthalpy shearing process and production of β-amyloid protein precipitate, (Αβ The formed senile plaque is a marker of neurodegenerative diseases;), and is a model of abnormal expression of secreted enzyme protein to observe the effect of abnormalities regulating secreted protein on Αβ production, and observation of prion protein and its product β ΑΡΡ-C terminal product (C99 and C83), the effects of beta-amyloid abnormalities on the development and progression of neurodegenerative diseases such as AD disease. In order to achieve this, the present inventors obtained a stable, heritable parental fruit fly strain through multi-generational hybridization using a GAL4/UAS transgenic system and a balanced lethal system, etc., using a Drosophila strain known in the prior art. The chromosome 2 of a Drosophila strain is ΑΡΡ/ΑΡΡ, the chromosome 3 is BACE/BACE; the chromosome 2 of another Drosophila strain is APP/APP, chromosome 3 is DPsn-BACE/DPsn-BACE. The present inventors finally obtained two Drosophila models suitable for studying neurodegenerative diseases such as AD, a chromosome of the Drosophila strain is APP/APP, chromosome 3 is BACE/BACE; APP is BACE The β APP-C end product of the cleavage product can be further cleaved by the endogenous DPsn of Drosophila to produce β-amyloid protein; the other chromosome of chromosome D is ΑΡΡ/ΑΡΡ, and the chromosome 3 is DPsn-BACE/ DPsn-BACE, a more significant pathological phenotype compared to the first Drosophila model. The inventors found through behavioral experiments and survival experiments that the two fruit fly models are significantly different from the previous AD fruit fly model in terms of survival and behavioral ability, as well as learning and memory ability (the pathological manifestations of this symptom are consistent with the symptoms of AD patients). .

 The previous Drosophila model was primarily a toxicity model that looked at the toxic effects of Aβ in the eyes of Drosophila. The APP/BACE fruit fly model of this application is significantly different from the previous model of Αβ overexpression (only the toxicity model). This model can simultaneously simulate the production process of Αβ and the toxic effects of Αβ, which can not only truly reflect the pathological features of AD. Moreover, effective behavioral indicators can be tested (previously the models expressed by the eyes cannot be tested for behavior), and these indicators can be used as an effective screening for indicators related to AD patients after drug treatment.

 In addition, studies have shown that cholinergic receptors play a very important role in the pathogenesis of AD. The former cholinergic expression model can be used as a drug screening and mechanism for targeting cholinergic neurons, and the latter is expressed in whole neurons. The model can reproduce the process of AD neurodegenerative diseases in Drosophila. The behavioral test using the Drosophila model of the present application is based on the expression of cholinergic neurons and whole neurons. As used herein, each gene marker is interpreted as follows:

 "Cyo" : located on chromosome 2 of Drosophila, is a winged marker of Drosophila, homozygous to death with this marker, heterozygous fruit flies appear as winged wings, cannot fly, fruit without the mark The flies appear as normal straight wings.

 "TM6B": located on chromosome 3 of Drosophila, the homozygous fruit fly with this marker appears to be dead, and the heterozygous fruit fly exhibits multiple and short tufts on both sides of the first thoracic segment, without The labeled fruit flies appear as two normal long bristles on either side of the first thoracic segment.

 " ΤΜΠ" : Located on chromosome 3 of Drosophila, the homozygous fruit fly with the mark appears to be dead, and the heterozygous fruit fly appears as a balanced rod with two short hairs and no fruit flies without the mark It is expressed as the end of the first thoracic section is the end of the balance bar without hair.

"B 1 " : located on chromosome 2 of Drosophila, the homozygous fruit fly with this mark appears dead Dead, heterozygous fruit flies appear as short hairs of the thoracic section, and fruit flies without this mark show normal thoracic bristles.

"Elavcl55": Drives GAL4 for expression of whole neurons, with a red-eye screening marker.

 "UAS-APP": APP expression gene under UAS with red eye screening marker.

 "UAS-BACE": BACE-expressing gene under UAS with red-eye screening marker. "X": A sex chromosome representing a fruit fly, the X chromosome.

 "y" : A sex chromosome representing a fruit fly, the Y chromosome.

 "+": It is attached to the back of the gene marker to indicate that the gene marker is present on the Drosophila chromosome; or it exists alone to represent the wild-type chromosome.

 It is linked to the back of the gene marker, indicating that the gene marker is not present on the Drosophila chromosome. The present invention also provides the use of the Drosophila model or a somatic cell or tissue thereof (such as brain tissue) for studying the interaction of a β-amyloid production process in a disease associated with β-amyloid overexpression; A medicament for screening for a disease associated with overexpression of β-amyloid by regulating (preferably reducing) the expression of a secretase.

 A study of the Drosophila model of the present invention found that the regulation of secreted enzymes can significantly alter the pathological progression of AD produced by the transgene Αβ. Therefore, the Drosophila model can be used to screen for novel target drugs for the treatment of AD diseases.

 Therefore, the present inventors significantly reduced the disease of AD by reducing or antagonizing the activity or expression of secretase protein in AD transgenic Drosophila, demonstrating the important role of the sputum cleavage process in AD progression and normal physiological functions. It plays a vital role in demonstrating its need as a new target for screening drugs.

 The Drosophila model can be prepared as described herein and can be assayed using conventional techniques in the art to determine if the prepared Drosophila has the desired genotype. For example, the genotype of the prepared Drosophila can be identified as described in the "Experimental Materials and Methods" section of this application. The methods described in the "Experimental Materials and Methods" section of this application can also be used to determine the expression of various enzymes in the screening methods described herein. Drug screening

After knowing the cleavage process of the cockroach and the role of the regulation of secreted protein in the disease associated with β-amyloid overexpression, various methods well known in the art can be used to screen for the regulation of the sputum shear process. A substance that can be used to ameliorate diseases associated with beta-amyloid overexpression. In a preferred aspect of the present invention, a method for screening for a potential substance for ameliorating a disease associated with β-amyloid overexpression, the method comprising: contacting a candidate substance with a system for expressing a secreted enzyme protein and a product protein , detecting the effect of the candidate substance on the secreted enzyme protein and the product protein; if the candidate substance can reduce the expression or activity of the secreted enzyme active protein, and alleviating the toxicity of the product, it indicates that the candidate substance can be used to improve β-amyloid protein Excessive expression of potential substances in related diseases. More preferably, if the candidate substance reduces the expression or activity of the secretase protein and does not completely inhibit its expression, it indicates that the candidate substance is a potential substance which is useful for improving the disease associated with β-amyloid overexpression, At the time of observation, a system in which a candidate substance is not added but expresses a secreted enzyme protein can be simultaneously set, so that the protein expression state can be more clearly analyzed.

 In the present invention, the system includes, but is not limited to, a solution system, a subcellular system, a cell system, a tissue system, an organ system, or an animal system. The system may contain prion protein and secretase protein for The candidate substance is added thereto to observe the influence of the candidate substance on the secreted enzyme protein; or, the system may contain both the secretase protein and the product Αβ protein for adding the candidate substance therein, and observing the candidate substance for the secreted enzyme The effect of protein and Αβ protein.

 In order to further select and determine a substance that is truly useful for ameliorating a disease associated with β-amyloid overexpression, the method further comprises: performing further cellular experiments and/or animal tests on the obtained potential substance.

In another method, the Drosophila model prepared using the methods described herein can be used as described above, and the screening can be screened by detecting changes or changes in behavior, survival, and/or memory capacity of the Drosophila model. A potential substance for improving the symptoms of neurodegenerative diseases caused by overexpression of β-amyloid. Generally, for behavioral changes or changes in the Drosophila model, if the crawling ability of AD Drosophila in the solvent control group is significantly improved by more than 5% (effective), 10% or more (better effect), 20% or more (effect) Excellent), the candidate substance is considered to be a potential substance for improving the symptoms of neurodegenerative diseases caused by overexpression of β-amyloid; for survival rate, if the survival rate of AD Drosophila is significantly higher than that of the solvent control group 5 More than % (good effect), more than 10% (better effect), more than 20% (excellent effect), the candidate substance is considered to be a potential for improving the symptoms of neurodegenerative diseases caused by β-amyloid overexpression. Substance; For learning ability, if the learning ability of AD Drosophila in the solvent control group is significantly improved by more than 5% (effective), 10% or more (better effect), 20% or more (excellent effect), the candidate is considered Substance is used to improve neurodegenerative damage caused by β-amyloid overexpression Potential substance for the symptoms of sexually transmitted diseases.

 The above single indicator can be tested, and any two or all of behavioral, survival, and learning abilities can be detected.

 These initially screened materials can constitute a screening library so that one can ultimately screen out substances that are indeed useful for improving diseases associated with β-amyloid overexpression.

 Accordingly, the present invention also encompasses a substance obtained by the screening method described, which is useful for ameliorating a disease associated with overexpression of β-amyloid. The main advantages of the invention are:

 (1) A fruit fly animal model for studying the interaction of Αβ and key enzyme proteins in the production process of neurodegenerative diseases (such as AD disease) and using them as screening targets. The main marker in neurodegenerative diseases, senile plaques formed by Αβ and its production process, can be used as research objects, and can also be used for the study of mechanisms and the screening of therapeutic drugs for unknown neurodegenerative diseases.

 (2) The animal model described is inexpensive to produce and has extremely reliable research value. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental conditions in which the specific conditions are not indicated in the following examples are generally carried out according to conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989) or William Sullivan et al. The conditions described in the Guide (New York: Cold Spring Harbor Laboratory Press), or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

 The Drosophila strain UAS-Α β ΐ used in the present invention is from the D.A丄 omas laboratory of the University of Cambridge, England, and the Drosophila strain UAS-APP, UAS-BACE is from the Drosophila Genetic and Molecular Biology Database (Indiana University).

Some of the Drosophila used by the inventors in the examples carry the GAL4-UAS expression system, wherein UAS-APP, UAS-BACE is used to transfer exogenous APP or BACE into progeny flies; GAL4 (ela _V cl55) Drosophila is used to express the gene of UAS in the elavcl55 Drosophila line in the daughter Drosophila neurons; GAL4 (cha) Drosophila is used to express the gene of UAS to the progeny Drosophila Cha Drosophila Drosophila strains in alkalinity neurons. The DB fruit fly (purchased from Shanghai Institute of Neurology, Chinese Academy of Sciences) used in the examples is a balanced transgenic fruit fly, and the balances with different screening markers (Cyo, Β1, ΤΜ6Β, ΤΜΠ) are inserted on chromosomes 2 and 3, Used for hybridization and stable inheritance of Drosophila.

 The invention will now be described in detail by way of specific examples. It should be understood that the invention is not limited to the specific embodiments. Experimental materials and experimental methods

 Transgenic fruit fly strain

• experiments with alpha] [beta transgenic flies model comprises: expressing transgenic flies low expression of wild-type Αβ42 (UAS-Αβΐ), Αβ 42 transgenic flies (UAS-AP2) expression and expression arctic mutant alpha] [beta transgenic fruit ₄₂ Fly (UAS-AParc).

• The APP/BACE transgenic Drosophila model includes: Transgenic Drosophila (UAS-APP) expressing APP protein, transgenic Drosophila (UAS-APP; BACE) co-expressing APP and BACE, co-transformed APP, BACE and Drosophila mutations Gene PSnlL235P transgenic fruit fly (UAS-APP; BACE; dPSnL235P). Two GAL4 strains of Drosophila were used in the experiment, namely elav ^el55- GAL4 expressed by whole neurons and cha-GAL4 expressed by cholinergic neurons. After mating, the first generation F1 was identified by genotype and protein expression and used for experiments. The fruit fly line UAS-AParc used in the article was from Dr. DALomas Laboratories, University of Cambridge, UK, and the fruit fly line UAS-A 2 was provided by Dr. Damian Crowther of the Cambridge University School of Medicine. See Figure 1 for a list of all the genetically modified fruit flies used in this article.

Table 1

果蝇分组及药物干预

Drosophila grouping and drug intervention

 • Male flies were incubated on adult day 1 and randomized: Αβΐ transgenic Drosophila group, Αβΐ transgenic Drosophila dosing group, Αβ2 transgenic Drosophila group, Αβ2 transgenic Drosophila dosing group, APP/BACE transgenic fruit fly group , P/VA & transgenic fruit fly dosing group, normal control group, normal fruit fly dosing group. Aricept Aricept (Tocris Cookson Ltd, UK), Hefei NTI (Sigma) were separately diluted and diluted to ΙΟμΜ and 30μΜ, and the Drosophila was continuously dosed in the drug intervention group. The non-drug intervention group was continuously dosed with solvent.

* Dosing method: Prepare 0.34mg/ml yeast sludge on the standard medium with the medicine, and change the medium every two days. The 0.55 mg/ml instant rice noodle medium was prepared with the drug, and the medium was changed every seven days. Primary reagent

 • Αβ murine monoclonal antibody 6E10 was purchased from Covance, Cat.#SIG-39320;

 • APP-CTF rabbit source polyclonal antibody was purchased from Sigma, Cat.# A8717;

 • Actin rabbit source polyclonal antibody purchased from Sigma, Cat.#A2066;

 · IRDye800CW conjugated goat anti-mouse IgG secondary antibody was purchased from Rockland.

Cat.#610- 131-121 ;

 • IRDye800CW coupled goat anti-rabbit IgG secondary antibody purchased from Rockland, Cat.# 611- 131-122;

 • FITC-conjugated goat anti-mouse IgG secondary antibody was purchased from Jackson ImmunoResearch, Cat. #l 15-095-146;

 • 2xPCR mix purchased from Tiangen Biochemical Technology Co., Ltd.

 • ELISA kit: Detection of human Αβ40 and Αβ42 were purchased from Biosource using the ELISA kit;

 • Naltrindole was purchased from Sigma;

 · Aricept is purchased from Tocris

 • γ-Secretase inhibitor L-685, 458 was purchased from Calbiochem;

 • Secretase Fluorescent Substrate was purchased from Calbiochem;

 • The plasmid extraction kit was purchased from Qiagen;

 Instruments and equipment

 · PCR amplification instrument was purchased from Bio-Rad;

 • Horizontal nucleic acid electrophoresis tank was purchased from Bio-Rad;

 • Desktop centrifuges were purchased from Ependoff;

 • Vertical electrophoresis system was purchased from Bio-Rad;

 • Gel imaging analysis system was purchased from Bio-Rad;

 · Low temperature benchtop refrigerated centrifuge purchased from Ependoff;

 • Odyssey far infrared analyzer;

 • DNA Concentration Analyzer was purchased from Thermo Scientific

 • Artificial climate box purchase Jiangnan Ningbo Instrument Factory;

• S88 Dual Output Exciter Electrical Exciter from Grass Technologies experimental method

 Drosophila cultivation and mating

 Amplify the parental fruit fly, and place a certain number of parents into the new medium depending on the scale of the experiment. After about 12 days, take the parental virgin flies for mating. The virgin fruit fly is white belly, larger than mature fruit flies and slightly transparent. On the 5th to 6th day after mating, the parental fruit fly is poured into a new tube to obtain more progeny fruit flies. The parental mating is about 12 days, and the F1 progeny flies are born. From the first day of birth, the offspring fruit flies are divided into 20 per tube, indicating the date of birth. All experimental transgenic flies were housed in an artificial climate chamber at 37 °C, 70% humidity, 12 hours during the day: 12 hours in the evening, and the Drosophila food was changed every two days. Genetically modified fruit fly genotype identification

 Drosophila genomic DNA was extracted according to standard methods. Take 10 genotypes of each genotype, and place 1 fruit fly in each EP tube, add 50μ1 lysate (10 mM Tris hydrochloric acid, pH 8.2, 1 mM EDTA, 25 mM sodium chloride, and 200 ug/ Ml protease K), fully ground and incubated in a 37 ° C water bath for 30 minutes. Then incubate at 95 °C for 5 minutes and centrifuge at 8,000 rpm for 15 min at 4 °C. Take the supernatant. The concentration was determined after dissolving the DNA.

Genotypic identification of Αβ transgenic Drosophila

 PCR identification of Αβ gene primers is:

 Abeta-PCR-F: GAC TGA CCA CTC GAC CAG GTT CTG (SEQ ID NO: 1) Abeta-PCR-R: CTT GTA AGT TGG ATT CTC ATA TCC G (SEQ ID NO: 2)

PCR reaction system:

 2xPCR mix buffer

 DNA template

 10 μΜ Abeta-PCR-F

 10 μΜ Abeta-PCR-R

Add water to make up 20 μl. PCR reaction conditions:

 94! : initial denaturation

Denaturation at 94 °C for 30 s,

60 °C renaturation for 60 s,

72. C amplification for 60 s,

Cycle 30 times.

APP/BACE transgenic Drosophila genotype identification

PCR identification of APP gene primers is:

 APP-F: CTTGTAGGTTGGATTTTCGTAGC (SEQ ID NO: 3) APP-R: ATGGTGGGCGGTGTTGTC (SEQ ID NO: 4)

PCR reaction system:

2xPCR mix buffer 10 μl,

DNA template 100 ng,

10 μΜ APP-F 0.5 μ1,

10 μΜ APP-R 0.5 μ1,

Add water to make up 20 μl.

PCR reaction conditions:

 94! : initial denaturation

Denaturation at 94 °C for 30 s,

60 °C renaturation for 60 s,

72. C amplification for 60 s,

Cycle 30 times.

PCR identification of PS 1 gene primers is:

dPSnL235P-F: CAGCTTTGTGGTGCTGGCTTCC (SEQ ID NO: 5) dPSnL235P-R: GGAAGCCAGCACCACAAAGCTG (SEQ ID NO: 6)

PCR reaction system:

 2xPCR mix buffer 10 μl,

 DNA template 100 ng,

 10 μΜ dPSnL235P-F 0.5 μ1,

 10 μΜ dPSnL235P-R 0.5 μ1,

 Add water to make up 20 μl. PCR reaction conditions:

 94! : initial denaturation

 Denaturation at 94 °C for 30 s,

 60 °C renaturation for 60 s,

 72. C amplification for 60 s,

 Cycle 30 times. Western blot

The adult fruit fly was anesthetized, the head was separated and collected, and about 100 heads of specific genotypes were placed in a 1.5 mL EP tube. Add 20 (^1^11? eight lysate (100111^/[13⁄45 117.4, 1 mM EGTA, 0.5 mM MgSO ₄ , 0.75 mM NaCl, 1% SDS, 0.02 mM NaF and cocktail protease inhibitor). After sonication at 4 Incubate for 40 minutes at °C, centrifuge for 1 hour at 24 000g, and collect the supernatant. Determine the protein concentration by BCA method. The protein sample is treated with 4χ loading buffer and then boiled at 95 °C for 5 minutes to obtain a thermostable protein lysate. The polyacrylamide gel electrophoresis was used to detect the expression of Αβ protein. The ΑΡΡβ murine monoclonal antibody 6E10-anti-treatment was used to detect the prion protein treated with APP-CTF rabbit polyclonal antibody. The BACE protein was detected by rabbit BACE polyclonal antibody.

For cell expression product extraction, the cell culture medium was discarded, and the cells were washed twice with pre-cooled PBS, and then lysis buffer (containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCK 5 mM EDTA, 10%) was added to the cells. Glycerin, 0.5 mg/ml bovine serum albumin, 1% Triton X-100 and protease inhibitor). The cells were harvested and lysed at 4 °C for 1 h. After the cells were lysed, they were centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was taken, the BCA protein was quantified, and the extracted protein product was detected by immunoblot hybridization. For immunoblotting, the protein bands were stimulated with far-infrared fluorescence by IRDyeSOOCW coupled secondary antibodies, obtained from the Odyssey Far Infrared Image System, and subsequently quantified on Scion Image software. Enzyme-linked immunosorbent assay for detection of Αβ levels

 Transgenic flies stably expressing Αβ and APP/BACE were sampled at appropriate ages for testing. The Αβ levels of transgenic fruit flies were determined using the human Αβ40 and Αβ42 enzyme-linked immunosorbent assay kits according to the manufacturer's method.

 The Drosophila brain was isolated by liquid nitrogen freezing and stored at -80 °C. 2% SDS solution was added to brain tissue before the experiment, thawed at room temperature, and then ultrasonically disrupted tissue. After the tissue was completely broken, it was centrifuged at 100 ° C for 1 h at 4 ° C for 1 h. The supernatant is a soluble protein component of SDS. The SDS soluble fraction was diluted and assayed using the human Αβ40 and Αβ42 enzyme-linked immunosorbent assay kits according to the manufacturer's method. Immunofluorescence and immunohistochemistry experiments

 Drosophila cultured to the appropriate age, anesthetized, placed in 4% PFA, pre-fixed for 15 minutes, removed and transferred to PBS, dissected under the microscope to expose the brain [30], fixed in 4% PFA for 30 minutes. . Wash 3 times with PBS for 30 minutes each time. The antigen was exposed to 70% formic acid for 30 minutes at room temperature. It was washed three times with a blocking solution of PBS-0.5% Triton X100-5% BSA for 30 minutes each time. The primary antibody was diluted with a blocking solution (6E10, 1:500) at 4 °C overnight. The primary antibody was removed, and the blocking solution was washed three times for 30 minutes each time, and the secondary antibody diluted with blocking solution (FITC-M, 1:100) was treated at room temperature for 2 hours. Remove the secondary antibody, wash the blocking solution three times, each time for 30 minutes, wash with PBS for 15 minutes. DAPI was treated at room temperature for 10 minutes and washed three times with PBS for 5 minutes each time. The Drosophila brain was transferred to a glass slide, mounted, and the sample was observed with a laser co-focus microscope (Leica TCS SP2 AOBS) and images were obtained.

 The area of the staining positive signal in the image was analyzed and counted using Image-Pro Plus 5.1 software. Each group of fruit flies takes 3-5 fruit flies, and each fruit fly takes 5-10 pieces of statistics, and the average value is taken as the group of fruit flies.

Hematoxylin-eosin tissue staining experiment

Drosophila is taken after anesthesia, 4% paraformaldehyde (4% PFA) fixed at 4 ° C overnight, washed with PBS buffer Three times, one hour each, in 1% in 30%, 50% ethanol, and stored in 70% ethanol at 4 °C. The fixed sample was dehydrated by gradient ethanol, transparent with xylene, embedded in paraffin at 54 °C, sliced conventionally, sliced to a thickness of 6 μm, attached to a cleaned glass slide, baked at 37 ° C overnight, and then collected in a slide cassette. Medium, sealed at 4 ° C.

 The sections were dewaxed by xylene, rehydrated with gradient ethanol, stained in hematoxylin for 30 seconds at room temperature, and the sections were quickly rinsed with water until the tissue sections turned blue and examined by microscopy. Separate with 0.1-0.5% hydrochloric acid ethanol for 1 to 2 seconds, then rinse the slices with running water until the slices turn blue, which is called blue. The sections were stained with gradient ethanol to 95% ethanol, stained in 0.5% eosin for 2 seconds or longer, and 95% ethanol was washed away from the residual color. If the color was too dark, it could be decolorized in a low concentration of ethanol and examined by microscopy. Dehydrated ethanol, transparent xylene, neutral gum seal. Under normal light microscope observation, the stained sections were preserved under room temperature drying conditions.

 3-5 fruit flies were taken from each group of fruit flies, and 5-10 pieces of each fruit fly were counted, and the average value was taken as the measurement value of the group of fruit flies. Drosophila survival rate statistics

 100 fruit flies were cultured in 5 food tubes in the number of 20 fruit flies per tube, and the culture temperature was maintained at 25 ° C and the humidity was 70%. The food tube is changed every 2-3 days to keep the food fresh. The number of fruit flies was counted daily and repeated 3 times. Drosophila crawling experiment

 Similarly, the number of 20 fruit flies per tube was 200 fish flies in 10 food tubes, and the culture temperature was maintained at 25 °C and the humidity was 70%, keeping the number of fruit flies larger than the number of fruit flies required for the experiment. Before the experiment, the fruit flies were placed in 10 clean empty tubes in 10 tubes/tubes and placed on the same horizontal bracket. During the experiment, the fruit fly was gently shaken to the bottom of the tube and recorded for 30 seconds. Repeat 6 times for each experiment. The tube was divided into 5 equal portions, and the number of fruit flies in each aliquot was recorded and the ratio was calculated at the 10th second. Drosophila learning and memory test

In the Drosophila olfactory-related learning and memory test, the test fruit fly was placed in a T-maze test device and given two different odors, trioctanol tetramethylcyclohexanol. During the training phase, one of the odors is coupled with the electric stimuli to give 60 seconds of training, rest for 45 seconds, and then pass to another odor without power. 60 seconds, rest for 30 seconds. In the exploratory index test phase, two odors were introduced at the same time, and the test fruit flies were given for 120 seconds to select, and then the electric shock exchange gas exchange was performed for a training and test, and the learning indexes of the two tests were respectively calculated. Value as a complete learning index. Eight complete learning indices were continuously measured and averaged as the learning index for the reorganized fruit fly. The learning index (PI value) is calculated as:

 Number of fruit flies making the right choice (+) - Number of fruit flies making the wrong choice (-)

Statistical analysis

 Experimental data is expressed as mean ± standard error. For behavioral experiments, data were analyzed by ANOVA and post-tested using Tukey post hoc tests. Survival analysis was performed using the kaplan-meier method in SPSS software. The survival rate was compared by log-rank test, and the difference was significant at p < 0.05. Example 1 : Stable Drosophila Drosophila System by Hybridization of Transgenic Drosophila

 In this example, the original transgenic Drosophila fruit flies were crossed with DB Drosophila to obtain the progeny flies with the transfer gene and the balancer selection marker, and the specific screening markers were screened to obtain the preserved lines (F2). ). The two generations of F2 generation Drosophila were crossed to obtain the second generation of fruit fly, and the two genetically stable impure and double transgenic fruit flies (F3) were obtained by simultaneously screening two different balances. The pure and double-transgenic Drosophila (F4) tool can be used to mate to produce a progeny Drosophila model.

 Two pairs of transgenic Drosophila (; F3) obtained by continuous hybridization were (APP/APP; BACE/BACE) and Π (ΑΡΡ/ΑΡΡ; DPsn/DPsn), which were obtained by crossing two kinds of tool transgenic Drosophila The progeny of the APP/secretase fruit fly model is APP/APP; DPsn-BACE/DPsn-BACE.

 The preparation method is as follows:

 A. Prepare F2 generation fruit flies separately:

(1) The UAS-APP strains of Drosophila (APP on chromosome 2) and UAS-BACE (BACE on chromosome 3) strains of Drosophila and DB strains of Drosophila (containing four kinds of balancers) were separated, and two F1s were isolated. Drosophila, a Drosophila APP on chromosome 2 APP/Cyo, chromosome 3 is +/TM6B; Another BRACE of Drosophila is BACE/TM6B on chromosome 3 and +/Cyo on chromosome 2; the two F1 Drosophila are backcrossed with DB fruit flies, respectively. The chromosomes of the F1 Drosophila are APP/Cyo; ΤΜ6Β/ΤΜΠ and B l/Cyo; BACE/TM6B, both contain 3 balancers. The above F1 Drosophila is crossed to obtain F2 generation Drosophila, the chromosome 2 of the F2 generation is APP/Cyo, and the chromosome 3 is BACE/TM6B; see Figure 1A, B;

 (2) The UAS-BACE/DPsn strains of Drosophila, the Drosophila No. 2 BACE on the chromosome, DPsn on chromosome 3, and the DB strain Drosophila (containing four kinds of balancers), respectively, separate the F1 generation of fruit flies, The F1 Drosophila APP is APP/Cyo on chromosome 2, and the chromosome 3 is BACE/TM6B; the F1 Drosophila is backcrossed with DB Drosophila, and the chromosome of the F1 Drosophila is Bl/Cyo. DPsn/TM6B, containing three balancers; this F1 is hybridized with F1 in (1) above to obtain F2 generation Drosophila, the F2 Drosophila APP is on chromosome 2 APP/Cyo, DPsn on chromosome 3 DPsn/TM6B; see figure ID;

 B. Prepare F3 generation fruit flies separately:

 (i) Hybrid F2 generation Drosophila obtained from (1) in step A and F2 generation Drosophila obtained in (2), and isolate F3 generation fruit fly, the chromosome 2 of the F3 generation Drosophila is APP/Cyo, 3 The chromosome is DPsn/BACE; some F3 generations of Drosophila may have chromosomal recombination from the parental and maternal chromosomes 3 during fertilization, and in this part of the F3 generation of fruit flies, chromosome 2 is APP/Cyo, chromosome 3 is DPsn-BACE/DPsn-BACE;

 (ϋ) All F3 Drosophila obtained in step (i) were crossed with DB Drosophila strain, and F3 Drosophila was isolated for PCR identification. The obtained F3 Drosophila Drosophila was on the chromosome 2 and APP/Cyo was on chromosome 2. , on chromosome 3 is DPsn-BACE/TM6B; see Figure IF;

 C. Prepare stable F4 generation fruit flies separately:

 (I) The two F2 generations of Drosophila obtained in (A) and (2) of step A are crossed to obtain F3 Drosophila, the chromosome 2 of the F3 Drosophila is APP/APP, and the chromosome 3 is BACE/ BACE; Another F3 generation of Drosophila chromosome 2 is APP/APP, and chromosome 3 is DPsn/DPsn.

 (II) Self-crossing F3 generation fruit flies positive in step B (ii) to obtain F4 generation fruit flies, the F4 generation Drosophila chromosome 2 is homozygous APP/APP, and chromosome 3 is homozygous DPsn- BACE/DPsn-BACE, see Figure 1G.

D. Prepare a double/triple traits fruit fly model separately: The F4 Drosophila obtained in (C) of the C step is hybridized with GAL4-cha to obtain the progeny Drosophila chromosome 2 as APP/+ and the chromosome 3 as BACE/+; the Drosophila in the cholinergic nerve The protein simultaneously expresses the substrate protein APP and β-secretase.

The F4 Drosophila obtained in (C) of the C step is crossed with GAL4-ela _{V to} obtain the progeny Drosophila chromosome 2 as APP/+ and the chromosome 3 as BACE/+; the Drosophila is in the whole neuron The substrate protein APP and β-secretase are simultaneously expressed.

 The F4 Drosophila obtained in (C) of the C step is hybridized with GAL4-cha to obtain the progeny Drosophila chromosome 2 as APP/+ and the chromosome 3 as BACE-DPsn/+; the Drosophila in choline The neurons can simultaneously express the substrate proteins APP and β-secretase and DPsn proteins.

The F4 Drosophila obtained in the C step (II) was crossed with GAL4-ela _{V to} obtain the progeny Drosophila chromosome 2 as APP/+ and the chromosome 3 as BACE-DPsn/+; Neurons simultaneously express the substrate proteins APP and β-secretase and DPsn proteins.

 The progeny Drosophila model produced above can be used for studies of neurodegenerative diseases and drug therapy. Example 2: Identification of Drosophila Model

 1. Human APP protein, β-secretase protein expressed in Drosophila

The C-terminus of human prion protein contains a fragment of β-amyloid, as shown in the black region of Figure 2, which is cleaved by β-secretase and γ-secretase in vivo to produce two forms of β-amyloid. , Αβ40 and Αβ42. In the transgenic Drosophila expressing UAS-APP, the inventors detected two glycosylated APPs at around 100 kDa using the ΑΡΡ gene C-terminal antibody against human prion protein (Fig. 2B, 11, 15 above) Lanes), after co-expression of human BACE protein, the expression of high molecular weight glycosylated APP was significantly reduced. It is indicated that in Drosophila, human BACE secretase is easy to shear high molecular weight APP. At the same time, the C-terminal fragment [β-C-terminal fragment (CTF)] of the sputum produced by β-secretase cleavage can be detected in Drosophila co-expressing APP and BACE (Fig. 2, above, lanes 1, 2 and 5) , 6). In the low-expression UAS-Αβ 1-1, UAS-A β 1-2 Drosophila, high-expression UAS-A I3 2 Drosophila neutralized the mutant UAS-A i arc ( E22G) In Drosophila, β-amyloid production can be detected in the brain of Drosophila with the antibody 6E10, which is a β-amyloid-terminal antibody. The molecular weight is about 3.5kDa (Fig. 2B, bottom, lane 1, 2, 3, 4卩 6, 7, 8, 9). Among them, the expression level of cholinergic neurons is lower, while that of whole neurons is higher. Meanwhile, the present inventors determined the levels of Αβ 40 and Αβ 42 in the brain tissue of Αβ-transgenic AD Drosophila by enzyme-linked immunosorbent assay. The present inventors have found that among the SDS soluble components, the AD transgenic Drosophila expressing Αβ expresses a higher Αβ 42 in the brain, and in the AD transgenic Drosophila brain of APP; BACE and APP; BACE-DPsn Both 40 β 40 and Α β 42 are generated (Fig. 2C).

 2. Detection of amyloid plaques detected in AD transgenic Drosophila with age In order to detect the production of Αβ amyloid plaques in the brain of AD Drosophila, the inventors performed a whole-area immunofluorescence experiment. . For the transgenic Drosophila (Αβ2) with high expression of Αβ, a large number of Αβ amyloid plaques were detected in the neural network region and the kenyon neuron cell body region at 20 days (Fig. 2D(b) arrow Show). For the transgenic Drosophila (Αβ 1 ) with low expression of Α β, only a small amount of fluorescence signal was detected during the 10-day life (Fig. 2D( )), and a part of the neural network region could be clearly detected by the 20-day life span. Α Formation of amyloid plaques (Fig. 2D (c.) Formation of significant A beta amyloid plaques in Drosophila expressing APP and secretase (APP; BACE and APP; BCE-DPsn) 2D(d) and (;e. However, such amyloid deposition is undetectable in wild-type Drosophila. Example 3: Verification of the deposition of amyloid caused by 剪切β by APP shearing by survival and behavioral experiments The neurotoxicity, regulation of β-secretase plays an important role in protecting neurons.

 Dissecting the pathological effects of human A{beta}40 and A{beta}42 in Drosophila: A potential model for Alzheimer's disease (PNAS, 2004, Zhong Yi) Et al.) - The specific scheme is as follows: The experimental fruit fly is as follows:

Wild type fruit fly (WT): elav ^el55 /CS (CS is a common wild type fruit fly, see the aforementioned PNAS, 2004, Zhong Yi et al.); AD fruit fly (Α β ): elav ^c155 /+ (y ; β 1/+, elav ^c155 /+(y) _; A β 2/+ (see the aforementioned PNAS, 2004, Zhong Yi et al.); AD/APP; BACE fruit fly (APP/+; BACE) /+): Example 1 Preparation; AD/APP; DPsn-BACE Drosophila (APP/+; DPsn-BACE/+): Example 1 Preparation.

Various fruit flies are cultured in the culture tube, and a small amount of food is added to the tube. The food includes 50g of corn flour, 20g of sugar, 15g of yeast powder, 10g of agar, another 600ml of water, and then 10-20ml. 15 tubes are dispensed into the culture tube. The fruit fly culture environment is 25 ° C and the humidity is 60 to 70%. Twenty-five Drosophila were cultured in one tube, five of which were used for survival experiments. The number of fruit flies was counted daily, the number of dead fruit flies was counted, and the viability index of Drosophila was observed by KM curve, as shown in Fig. 3A and B. The results indicate that the cleavage process of APP protein in Drosophila can produce toxic amyloid deposits, which can significantly shorten the lifespan of this AD fruit fly. Another 6 tube flies were used to calculate the crawling ability of Drosophila. The experimental method was as follows: In the dark, 20 flies per tube were transferred into empty tubes, each tube was 9.5 cm long and 2.5 cm in diameter. The fruit fly shakes to the bottom of the tube, uses the reverse ground instinct of the fruit fly to make the fruit fly crawl upwards, divides the test tube into 5 grids, counts the number of fruit flies per cell after 10 seconds, and uses the total number of fruit flies to crawl. Divide by time to calculate the average speed of each fruit fly, recorded as its crawling index, use the line chart to calculate the crawling index of Drosophila, as shown in Figure 3C, with the increase of age, the mobility of AD Drosophila significantly decreased . However, AD/APP; BACE flies fed beta secretase inhibitors can significantly prolong the lifespan of AD Drosophila, and the results show that reducing BACE protein activity can significantly improve the survival time of AD Drosophila. Summarizing the above results, we can conclude that AD/APP; B ACE and AD/APP; BACE-DPsn's fruit flies show significant age-dependent degenerative lesions, thus showing defects in behavioral ability, while inhibiting secreted enzymes Activity can alleviate its symptoms.

In order to verify the above phenotype as an effective method and route for drug screening against Αβ toxicity and targeted regulation of Αβ production, the present inventors added a β-secretase inhibitor to the Drosophila-fed medium [Sinha, S ., et al, Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature, 1999. 402(6761): p. 537-40; Dovey, HF, et al., Functional gamma-secretase inhibitors reduce beta-amyloid Peptide levels in brain. J Neurochem, 2001. 76(1): p. 173-81]. Infecting APP/BACE transgenic flies with ΙΟηΜ and 30 nM β-secretase inhibitors, the inventors found that β-secretase inhibitors can significantly prolong the lifespan of AD fruit flies and prolonged as the feeding dose increases The effect is more pronounced (η=100, Ρ<0.01). The inhibitors of β-secretase of 3ηΜ and ΙΟηΜ extended the lifespan of Drosophila in the solvent control group for 3 days and 9 days, respectively (Fig. 3D shows dark blue and light blue lines). Inhibitors of β-secretase treated Drosophila expressing Aβ did not change their survival time (Fig. 3Ε). These results suggest that β-secretase inhibitors do not directly act on Aβ to alleviate its toxicity, but inhibit the activity of β-secretase (BACE), which regulates key enzymes in the production of Aβ, thereby alleviating Aβ. The toxic effects produced. At the same time, this result suggests that transgenic fruit flies using APP/BACE can be used not only as a way to study the development of AD and The experimental animal model of the mechanism, and can be a good animal model for anti-AD drug screening. Example 4: Using a Drosophila Disease Model for Drug Screening

 The disease model is characterized by sub-genus Drosophila exhibiting significant age-dependent degenerative lesions, thereby exhibiting behavioral deficiencies (proven in Example 3). In this example, screening of effective drugs using each of the aforementioned Drosophila strains can alleviate the progression of age-dependent degenerative diseases and inhibit damage caused by neurotoxicity.

 1. Arison can significantly extend the lifespan of AD fruit flies and alleviate the deficits in the ability of AD fruit flies.

 In the same way, other possible anti-AD pathological drugs were validated and screened on the AD Drosophila model. The inventor added Anritsu to the culture medium of the fruit fly, and Anritsu is an inhibitor of acetylcholinesterase, which is now widely used in the clinical use of mild to moderate senile dementia. Therefore, the present inventors fed two transgenic male Drosophila models specifically expressing Αβ or APP/BACE to cholinergic neurons, respectively, and feeding ΙΟμΜ and 30 μΜ of Aricept, and then the inventors found that Aricept can significantly prolong Αβ low expression transgene. The lifespan of Drosophila, and with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). ΙΟμΜ and 30μΜ of Arison extended the lifespan of the Drosophila control group for 5 days and 6 days, respectively (Fig. 4Α (a) The dark blue and light blue lines on the left show survival curves, and the middle is the statistical results of survival curves). Similarly, ΙΟμΜ and 30μΜ of Arimoto can significantly alleviate the deficiencies in the ability of Αβ to express transgenic fruit flies, as shown in Figure 4 (a).

 Anritsu can significantly prolong the lifespan of Αβ-expressing transgenic fruit flies, and with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). ΙΟμΜ and 30μΜ of Arison extended the lifespan of the Drosophila control group for 6 days and 7 days, respectively (Fig. 4Α (b) The dark blue and light blue lines on the left show survival curves, and the middle is the statistical results of survival curves). Similarly, ΙΟμΜ and 30 μΜ of Aricept can significantly alleviate the deficiencies in the mobility of Αβ-expressing transgenic fruit flies, as shown in the right panel of Figure 4A (b).

 For APP/BACE transgenic fruit flies, the effect of Arison was not significant (n=100). but,

ΙΟμΜ and 30μΜ of Arimoto can significantly alleviate the deficit of APP/BACE transgenic fruit flies, as shown in the right panel of Figure 4A (c).

The treatment of wild type flies in Drosophila did not alter their survival (Fig. 4A(d)). The above results suggest that ΙΟμΜ and 30μΜ of Anritsu relieve the toxic effects of Aβ through a mechanism. 2. The two transgenic male Drosophila models with full neuron-specific expression of Aβ or APP/BACE were fed with ΙΟμΜ and 30 μΜ of Arison, respectively. The inventors also found that Aricept can significantly prolong the lifespan of Αβ low-expression transgenic fruit flies. And with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). ΙΟμΜ and 30μΜ of Arimoto extended the lifespan of the Drosophila control group for 6 days and 7 days, respectively (Fig. 4Β( ) shows the survival curve in the dark blue and light blue lines on the left, and the statistical results of the survival curve in the middle). Similarly, ΙΟμΜ and 30 μΜ of Aricept can significantly alleviate the deficiencies in the mobility of Αβ low-expression transgenic Drosophila, as shown in the right panel of Figure 4B(a).

 Anritsu can significantly prolong the lifespan of Αβ-expressing transgenic fruit flies, and with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). ΙΟμΜ and 30 μΜ of Anritsu extended the lifespan of Drosophila in the solvent control group for 4 days and 6 days, respectively (Fig. 4B(b) The dark blue and light blue lines on the left show the survival curve, and the middle is the statistical result of the survival curve). Although the effect of prolonging life is not as good as that of cholinergic neurons, the expression of exogenous proteins in whole neurons is higher and produces a stronger toxic effect. The efficacy of Aricept is not completely resistant to this toxicity. . In addition, ΙΟμΜ and 30μΜ of Arimoto can significantly alleviate the deficiencies in the ability of Αβ to express transgenic fruit flies, as shown in the right panel of Figure 4B(b).

 For APP/BACE transgenic fruit flies, the effect of Arison was also not significant (n=100). However, ΙΟμΜ and 30μΜ of Arimoto can significantly alleviate the deficit of APP/BACE transgenic fruit flies, as shown in the right panel of Figure 4B(c).

 The treatment of wild type flies in Drosophila did not alter their survival (Fig. 4B(d)). The above results suggest that ΙΟμΜ and 30μΜ of Arimoto may directly relieve the toxicity of Aβ through a certain mechanism and play a neuroprotective role.

3. ΝΤΙ can significantly extend the lifespan of AD fruit flies and alleviate the defects in AD Drosophila's ability to act. Recently, the inventors' research group found that β-AR and sputum-secretase need to form a complex with δ opioid receptors, and together promote amyloid production. The study found that the use of the drug Naltrindole (NTI) against S opioid receptors can effectively alleviate amyloid production, in the process, no side effects, the inventors believe that this will be a potential for Alzheimer's disease Treatment target. For this mechanism, the inventors hope to further verify in vivo in AD transgenic Drosophila, and apply AD transgene Drosophila, the effects of opioid receptor antagonists on downstream Αβ toxicity can also be studied.

 Similarly, the present inventors fed two transgenic male Drosophila models specifically expressing Αβ or APP/BACE to cholinergic neurons, respectively, and feeding ΙΟμΜ and 30 μΜ of ΝΤΙ, and then the inventors found that NTI can significantly prolong Αβ low expression transgenic fruit flies. The lifespan, and with the increase in feeding dose, the prolongation effect is more obvious (η = 100, Ρ < 0.01). ΙΟμΜ and 30 μΜ of Arison extended the fruit fly life of the solvent control group for 4 days and 5 days, respectively (Fig. 4C (a) The left red and red lines show survival curves, and the middle is the statistical results of the survival curve). Similarly, ΙΟμΜ and 30μΜ of NTI can significantly alleviate the deficiencies in the ability of Αβ to express transgenic Drosophila, as shown in the right panel of Figure 4C( ).

 NTI can significantly prolong the lifespan of Αβ-expressing transgenic fruit flies, and with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). The TIμΜ and 30μΜ NTI extended the lifespan of the Drosophila in the solvent control group for 3 days and 5 days, respectively (Fig. 4C(b) left dark red and light red lines show survival curves, and middle is the statistical result of survival curve). Similarly, ΙΟμΜ and 30μΜ of NTI can significantly alleviate the deficiencies in the mobility of Αβ-expressing transgenic fruit flies, as shown in the right panel of Figure 4C(b).

 For APP/BACE transgenic fruit flies, the effect of NTI was more pronounced (n=100, P<0.01). ΟμΜ and 30 μΜ of NTI extended the lifespan of the Drosophila control group for 3 days and 5 days, respectively, as shown in Figure 4C. Similarly, ΙΟμΜ and 30 μΜ of NTI can significantly alleviate the deficit in the performance of APP/BACE transgenic fruit flies, as shown in the right panel of Figure 4C(c).

 The same was obtained, and treatment of wild-type Drosophila fruit flies with NTI did not change their survival time (4C(d)). These results suggest that ΙΟμΜ and 30μΜ of NTI can alleviate the toxic effects of downstream Aβ through some mechanism, and may intervene and regulate from the sputum shearing process, thus alleviating the pathological symptoms of APP/BACE transgenic Drosophila.

4. Similarly, the present inventors fed two transgenic male Drosophila models specifically expressing Αβ or APP/BACE to 神经μΜ and 30 μΜ of ΝΤΙ, respectively, and the inventors found that NTI can significantly prolong Αβ low expression of transgenic fruit flies. Lifespan, and with the increase in feeding dose, the prolongation effect is more obvious (η = 100, Ρ < 0.01). ΙΟμΜ and 30μΜ of NTI were extended in the solvent control group for Drosophila lifespans of 4 days and 3 days, respectively. Figure 4D ( ) The left red and red lines show survival curves, and the middle is the statistical results of survival curves). Similarly, ΙΟμΜ and 30μΜ of Arimoto can significantly alleviate the deficiencies in the mobility of Αβ low-expression transgenic Drosophila, as shown in the right panel of Figure 4D(a). NTI can significantly prolong the life span of transgenic Drosophila with high expression of Αβ, and with the increase of feeding dose, the prolongation effect is more obvious (η=100, Ρ<0.01). The TIμΜ and 30μΜ NTI extended the lifespan of the Drosophila in the solvent control group for 3 days and 4 days, respectively (Fig. 4D(b) left dark red and light red lines showed survival curves, and the middle was statistical results of survival curves). Similarly, ΙΟμΜ and 30 μΜ of NTI can significantly alleviate the deficiencies in the mobility of Αβ-expressing transgenic fruit flies, as shown in the right panel of Figure 4D(b).

 Similarly, for APP/BACE transgenic flies, the effect of NTI was more pronounced (n=100, Ρ<0.01). ΟμΜ and 30 μΜ of NTI extended the lifespan of the Drosophila control group for 5 days and 4 days, respectively, as shown in Figure 4D. Similarly, ΙΟμΜ and 30μΜ of ΝΤΙ can significantly alleviate the defect in the ability of APP/BACE transgenic genus, as shown in the right panel of Figure 4D(c).

 The same treatment, using NTI to treat wild-type Drosophila fruit flies did not change their survival time

(Fig. 4D(d)).

5. Arison and NTI can significantly improve the decline in learning ability of AD fruit flies

 The test method for learning and memory related to olfactory sense in Drosophila is as follows: The test fruit fly is placed in a T-maze test device and given two different odors, trioctanol tetramethylcyclohexanol. During the training phase, one of the odors is coupled with the electric stimuli for 60 seconds of training, rest for 45 seconds, and the other odor is not energized. The stimuli are coupled for 60 seconds and rest for 30 seconds. In the exploratory index test phase, two odors were introduced at the same time, and the test fruit flies were given a choice of 120 seconds to select, and then the electric stimulation coupled gas exchange was carried out for a training and test, and the learning indices of the two tests were respectively calculated. Value as a complete learning index. Eight complete learning indices were continuously measured and averaged as the learning index for the reconstituted fruit fly. The learning index (PI value) is calculated as described above.

 The experimental results are shown in Figure 4E. The results showed that both Anritsu and NTI were effective against β-amyloid toxicity, and the effect of Aricept was more obvious. For AD/APP; BACE's Drosophila model was more significant than the mechanism of action of Arison. It alleviates the decline of the learning index and effectively enhances the learning ability of AD fruit flies.

6. The neuroprotective effect of Aricept and sputum is not achieved by directly reducing the amount of Αβ. In this example, the inventors also detected the Drosophila (Aβ 2 ) brain in the drug-administered group by immunoblotting. There is no reduction in the internal Αβ, see Figure 5Α. Immunofluorescence showed no beta-amyloid deposits There is a significant decrease, indicating that Anritsu and NTI did not directly remove the already generated Αβ (Fig. 5Β). At the same time, the present inventors also performed a hematoxylin-eosin staining assay to detect plaques caused by neuronal apoptosis in the brain of AD transgenic Drosophila. At the 25-day lifespan of AD Drosophila, a large number of plaques were clearly detected in the neural network region and the Kenyon neuronal cell region (shown by the arrow in Figure 5C). The reduction and reduction of plaque in the brain of Drosophila treated by Arison and NTI suggests that the toxicity caused by β-amyloid deposition may be alleviated by other pathways rather than direct action, which plays a neuroprotective role. In summary, by regulating the endogenous expression or activity of the secreted enzyme protein, the pathological symptoms of Drosophila AD caused by β-amyloid toxicity can be effectively alleviated. This confirmed the important role of prion protein and its shear process in pathological and physiological processes, further illustrating the importance and feasibility of key proteins in the process of prion cleavage dynamics as a therapeutic target for AD disease. At the same time, the behavioral index and pathological detection index of the disease model can be used as a direct response to the drug treatment effect, providing an effective and quick tool for the disease-related process and treatment effect. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art after the above-described teachings of the present invention.

Claims

A method of preparing a fruit fly model, the method comprising: (1) The UAS-APP strain Drosophila is crossed with the DB strain Drosophila, and the Drosophila fruit flies are backcrossed with DB to obtain Drosophila with chromosome 2 as APP/Cyo and chromosome 3 as ΤΜ6Β/ΤΜΠ;  (2) The UAS-BACE strain Drosophila is crossed with the DB strain Drosophila, and the Drosophila fruit flies are backcrossed with DB to obtain Drosophila with chromosome 2 as Bl/Cyo and chromosome 3 as BACE/TM6B;  (3) Crossing the Drosophila lines obtained in steps (1) and (2), and then self-crossing the resulting Drosophila chromosome 2 as APP/Cyo and chromosome 3 as BACE/TM6B to obtain chromosome 2 It is a homozygous APP/APP, and the chromosome 3 is a homozygous BACE/BACE stable genetic flies;  (4) The UAS-BACE; DPsn strain Drosophila was crossed with the DB strain of Drosophila, and then backcrossed with the DB strain to obtain Drosophila with chromosome 2 as Bl/Cyo and chromosome 3 as DPsn/TM6B;  (5) The Drosophila obtained in the step (1) is crossed with the Drosophila obtained in the step (4) to obtain a fruit fly with the chromosome 2 as APP/Cyo and the chromosome 3 as DPsn/TM6B;  (6) The Drosophila obtained in the step (5) is hybridized with the chromosome 2 obtained in the step (3) as the APP/Cyo, and the chromosome 3 is the BACE/TM6B progeny, and the chromosome 2 is APP/Cyo. Chromosome recombination of chromosome 3, DPsn-BACE/DPsn-BACE, which exhibits three traits of Drosophila;  (7) The Drosophila obtained in the step (6) is crossed with DB to obtain a fruit fly with chromosome 2 as APP/Cyo and chromosome 3 as DPsn-BACE/TM6B;  (8) Self-crossing the fruit fly obtained in step (7) to obtain a stable genetic inherited flies from chromosome 2 to homozygous APP/APP and chromosome 3 to homozygous DPsn-BACE/DPsn-BACE. Fly model.  2. The method according to claim 1, wherein the method further comprises:  (9) The Drosophila obtained in the step (8) is crossed with the DB, and the progeny Drosophila is crossed back with +/+; TM6B/TMII, and the chromosome 2 of the obtained fruit fly is +/+ The chromosome 3 is DPsn-BACE/DPsn-BACE; the Drosophila expresses only β-secretase and presenilin but does not express substrate peptone.  3. The method according to claim 1, wherein the method further comprises: (10) The fruit fly obtained in step (8) is crossed with GAL4 (cha) to obtain a cholinergic a progeny that specifically expresses a target protein within a cell; or (11) The Drosophila obtained in the step (8) is hybridized with GAL4 (el _av ) to obtain progeny which can specifically express the target protein in the whole neuron.  A Drosophila somatic cell or tissue characterized in that said tissue co-expresses APP protein and beta secretase (BACE) and does not overexpress or overexpress Drosophila presenilin protein (Dpsn).  The Drosophila somatic cell or tissue according to claim 4, wherein the tissue is selected from the nerve tissue of Drosophila.  6. Use of a fruit fly prepared by the method of any one of claims 1 to 3, or a Drosophila somatic cell or tissue according to claim 4 or 5, characterized in that  (a) for studying the interaction of the substrate APP and its cleavage enzymes β-secretase and γ-secretase; (b) for the study of the role of the intermediate splicing product βΑΡΡ-CTF (C99) and the pathological process of the final product of β-molar protein overexpression-related diseases;  (c) for studying the correlation between prion cleavage processes and diseases associated with β-amyloid overexpression;  (d) To study the pathological process of APP protein cleavage and β-amyloid overexpression in specific neuronal tissues;  (e) a drug for screening for the interaction of β-secretase, γ-secretase and/or β-amyloid in the progression of a disease associated with β-amyloid overexpression;  (f) a drug for screening for a disease associated with β-amyloid overexpression by regulating the expression of β-secretase and/or γ-secretase and a target protein interacting therewith;  (g) A medicament for the preparation of a disease associated with the treatment of β-amyloid overexpression.  The use according to claim 6, wherein the β-amyloid overexpression-related disease is a neurodegenerative disease.  The use according to claim 7, wherein the neurodegenerative disease is Alzheimer's disease.  9. A method of screening for potential substances that ameliorate a disease associated with β-amyloid overexpression, the method comprising:  (1) contacting the candidate substance with a system expressing a β-secretase protein or a γ-secretase component protein; (2) detecting the effect of the candidate substance on the β-secretase protein or the γ-secretase component protein; Wherein, if the candidate substance can reduce the expression or activity of the β-secretase protein or the γ-secretase component protein, it indicates that the candidate substance is a potential substance which can be used to improve the disease associated with β-amyloid overexpression.  10. A method of screening for potential substances that ameliorate a disease associated with β-amyloid overexpression, characterized in that the method comprises:  A method for preparing a fruit fly according to the method of any one of claims 1 to 3;  Giving the Drosophila model candidate substance; and  Detecting the effect of the candidate substance on the β-secretase protein or the γ-secretase component protein in the Drosophila model, or detecting the behavior of the Drosophila model;  Wherein, if the candidate substance can reduce the expression or activity of the β-secretase protein or the γ-secretase component protein in the Drosophila model, it indicates that the candidate substance is useful for improving the β-amyloid overexpression related diseases. Potential substance; or, if the candidate substance alters the behavior, survival rate, and/or memory capacity of the Drosophila model, indicating that the candidate substance is useful for improving neurodegenerative diseases caused by β-amyloid overexpression The underlying substance of the symptoms.