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WO2009030086A1 - Fonctions et utilisations d'un inhibiteur de croissance dérivé des cellules stromales de la moelle osseuse humaine - Google Patents

Fonctions et utilisations d'un inhibiteur de croissance dérivé des cellules stromales de la moelle osseuse humaine Download PDF

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Publication number
WO2009030086A1
WO2009030086A1 PCT/CN2007/070626 CN2007070626W WO2009030086A1 WO 2009030086 A1 WO2009030086 A1 WO 2009030086A1 CN 2007070626 W CN2007070626 W CN 2007070626W WO 2009030086 A1 WO2009030086 A1 WO 2009030086A1
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Prior art keywords
bdgi
polypeptide
cells
tumor
human
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Chinese (zh)
Inventor
Xuetao Cao
Nan Li
Dajing Xia
Tao Wang
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention belongs to the fields of biotechnology and medicine, and in particular, the present invention relates to a novel human growth inhibitory protein, Bone Marrow Stromal Cell-derived Growth Inhibitor (BDGI), and a polypeptide encoding the same.
  • BDGI Bone Marrow Stromal Cell-derived Growth Inhibitor
  • Tumors are diseases that seriously endanger human health. According to WHO statistics, the average number of people who die of malignant tumors per year among the more than 6 billion people in the world is 7.2 million, and the number of new cases is 8.9 million, and the number is increasing year by year. This is also the case in the domestic situation. The composition ratio of malignant tumors in the cause of death has increased from 12.6% to 17.9% in the past 20 years. As a result, governments, research institutions, and pharmaceutical companies have long placed a high priority on cancer research and anti-cancer drugs.
  • the disorder of cell growth regulation is the main cause of tumorigenesis, which is caused by the imbalance of positive regulatory signals that promote cell growth and proliferation, prevent terminal differentiation, and negative regulation signals that promote cell maturation and apoptosis.
  • the mechanism involves activation of proto-oncogenes, inactivation of anti-oncogenes, persistence of growth and proliferation signals, and abnormal cell cycle. Therefore, studying the molecular mechanism of tumor formation, occurrence and development at multiple levels is conducive to re-establishing the dynamic balance between positive and negative regulatory signals at the molecular level, in order to achieve the desired therapeutic effect.
  • Apoptosis is a gradual death of genes that are actively regulated by specific genes in specific time and space. It plays an important role in tumorigenesis, development, tumor therapy, embryonic development, immune response, nervous system development, and tissue cell metabolism. effect. Apoptosis is an important component of the cell monitoring system. If it is abnormal, it will cause the cells that should be apoptotic to "illegally" survive, resulting in uncontrolled proliferation of cells, resulting in highly degraded tumor cells. In the process of cell carcinogenesis, many apoptotic activating genes are blocked, and the function of apoptosis-inhibiting genes is enhanced. It has been found that many tumors are caused by obstruction of apoptosis, such as lymphoma, breast cancer, prostate cancer, ovarian cancer, and chronic leukemia.
  • Growth inhibitory proteins having a cell growth inhibiting action are generally low molecular weight polypeptides or glycoprotein molecules which are capable of inhibiting cell growth in vivo by binding to corresponding receptors on the cell surface.
  • Growth inhibitors such as transforming growth factor bl (TGF-bl), mammastatin (mammastatin neuregulin (NDF), mammary gland growth inhibitory factor (MDGI), and MDGI-related genes have been reported in the literature in the last decade. MRG) and the like have potential anti-mammary tumors and other tumor formation effects.
  • TGF-bl growth inhibitors
  • mammastatin is a peptide derived from the conditional medium of normal mammary epithelial cells, which inhibits normal and transformed cultures in vitro. DNA synthesis of mammary epithelial cells; MDGI and MRG can stimulate the differentiation of breast cancer epithelial cells in vitro, cause local cell proliferation inhibition, and inhibit tumor growth in nude mice.
  • BDGI is a novel growth inhibitory factor obtained from large-scale random sequencing of BMSC cDNA gene library. It has a total length of 1766 bp and contains a complete open reading frame of 1434 bp, which encodes 477 amino acids. The gene is located at 16q24. 1. This gene has been shown to inhibit the growth of breast cancer cell line MCF-7.
  • a human bone marrow stromal cell-derived growth inhibitory factor, BDGI polypeptide or a coding sequence thereof for use in the preparation of a composition for inhibiting tumor cell migration.
  • the BDGI polypeptide is selected from the group consisting of
  • the composition is also for promoting tumor cell apoptosis. In another embodiment of the invention, the composition is a pharmaceutical composition.
  • the composition contains a pharmaceutically acceptable carrier, diluent or excipient, and 0.001 to 99.99% by weight of a human BDGI polypeptide or a coding sequence thereof, to which the composition The total weight is based on the basis.
  • the human BDGI polypeptide or a coding sequence thereof is contained in an amount of from 0.01 to 95% by weight, preferably from 0.1 to 90% by weight, more preferably from 0.5 to 80% by weight.
  • the tumor cells are selected from the group consisting of breast cancer cells and lung cancer cells.
  • the pharmaceutical composition further comprises an additional anti-cancer drug.
  • the coding sequence is selected from the group consisting of:
  • a method of screening a human bone marrow stromal cell-derived growth inhibitory factor, an agonist or inhibitor of a BDGI polypeptide, in vitro comprising:
  • control group is a culture system of a tumor cell expressing a BDGI polypeptide or a culture system of a tumor cell to which a BDGI polypeptide is added
  • test group is added with a test substance a culture system of a tumor cell expressing a BDGI polypeptide, or a culture system of a tumor cell to which a test substance and a BDGI polypeptide are added;
  • the migration ability of the tumor cells in the test group is increased compared with the control group, indicating that the test substance is an inhibitor of a potential BDGI polypeptide; wherein, in comparison with the control group, the migration of the tumor cells in the test group A decrease in capacity indicates that the test substance is an agonist of a potential BDGI polypeptide.
  • the culture system of the tumor cell expressing the BDGI polypeptide or the culture system of the tumor cell to which the BDGI polypeptide is added is a culture system of MCF-7 cells overexpressing the BDGI polypeptide.
  • the method is for screening for an inhibitor, and in step (b), further comprising comparing migration of the tumor cells in the test group to migration of a positive control group
  • the positive control group was a culture system of tumor cells expressing a BDGI polypeptide to which an anti-BDGI antibody was added, or a culture system to which tumor cells to which an anti-BDGI antibody and a BDGI polypeptide were added.
  • Figure 1 shows the Western blot analysis of human BDGI eukaryotic recombinant expression vector transfected human tumor cells expressing BDGI protein.
  • Figure 1A Human MCF-7 breast cancer cells;
  • Figure 1B Human A549 lung cancer cells.
  • Figure 2 shows the inhibition of tumor cell proliferation by overexpression of human BDGI protein of the present invention.
  • Figure 2A AMCF-7 breast cancer cells;
  • Figure 2B Human A549 lung cancer cells.
  • Figure 3 shows the inhibition of tumor cell colony formation by human BDGI protein overexpression.
  • Figure 4 shows the inhibition of tumor cell migration by human BDGI protein overexpression.
  • Figure 4A Average number of cells migrating per field of view;
  • Figure 4B Average distance of cell migration per field of view.
  • Figure 5 is the induction of tumor cell apoptosis by overexpression of human BDGI protein.
  • Figure 5A Annexin V/PI staining results;
  • Figure 5B Scanning electron micrographs. detailed description
  • the present inventors conducted extensive and in-depth research on the anticancer effect of the growth inhibitory factor-Bone marrow stromal cell-derived growth inhibitory factor BDGI obtained from large-scale random sequencing of the BMSC cDNA gene library, and confirmed in various aspects through various experiments.
  • the BDGI polypeptide has a function of inhibiting tumor cell migration and promoting apoptosis of tumor cells, and is effective for inhibiting and treating tumors, thereby completing the present invention.
  • overexpression of the BDGI protein of the present invention can significantly inhibit the migration of human tumor cells (e.g., MCF-7 breast cancer cells and A549 lung cancer cells), and reduce the colony formation rate of tumor cells in soft agar.
  • overexpression of BDGI protein can also induce tumor cell apoptosis.
  • the inhibition of tumor cell migration and induction of tumor cell apoptosis by BDGI protein suggests that BDGI protein is an important growth inhibitory factor in malignant transformation of normal cells, and it is involved in the occurrence, development and metastasis of tumors.
  • BDGI is involved in tumorigenesis and biological behavior, and can regulate a variety of physiological and pathological activities, and thus has important development and application value in immunodiagnosis and immunotherapy in various fields such as anti-tumor and immune function regulation.
  • the invention also provides the use of a coding sequence for a BDGI polypeptide encoding a polypeptide having the BDGI amino acid sequence set forth in SEQ ID NO: 2, for use in inhibiting and treating a tumor.
  • the polynucleotide of the present invention is isolated from a human bone marrow stromal cell cDNA library. Its sequence is shown in SEQ ID NO: 1: It contains a polynucleotide sequence of 1766 bases in full length and its open reading frame is located at 147-14580, encoding a human BDGI protein of 477 amino acids in length (SEQ ID). NO: 2).
  • the isolated polynucleotide produces a BDGI polypeptide by encoding to achieve an effect of inhibiting and treating a tumor.
  • BDGI protein or peptide
  • BDGI polypeptide or “bone marrow stromal cell-derived growth inhibitory factor BDGI” are used interchangeably and refer to a growth inhibitory factor BDGI amino acid sequence derived from human bone marrow stromal cells (SEQ ID N0 : 2) A protein or polypeptide, a conservative variant polypeptide thereof, an active fragment thereof, or an active derivative thereof.
  • the "BDGI polypeptide” is selected from the group consisting of:
  • BDGI protein includes a variant form of the sequence of SEQ ID NO: 2 which has the same function as the human BDGI protein.
  • variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions And/or substitution, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • a number usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions And/or substitution, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • amino acids usually within 20
  • amino acids of similar or similar properties the function of the protein is generally not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to human BDGI DNA under high or low stringency conditions And a polypeptide or protein obtained using an antiserum against an anti-human BDGI polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising a human BDGI polypeptide or a fragment thereof. In addition to nearly full length polypeptides, the invention also includes soluble fragments of human BDGI polypeptides.
  • the fragment has at least about 10 contiguous amino acids of the human BDGI polypeptide sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimally at least about 100 consecutive amino acids.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the native human BDGI protein of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic Ig
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in the natural state of living cells It is not isolated and purified, but the same polynucleotide or polypeptide, if separated from other substances present in the natural state, is isolated and purified.
  • isolated BDGI protein or polypeptide means that the BDGI polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art will be able to purify the BDGI protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • polypeptides of the invention may also be used in conjunction with proteins of molecular weight such as BSA to form polypeptide conjugates.
  • the conjugate consists of a polypeptide, a crosslinker, and BSA, wherein the crosslinker is preferably glutaraldehyde, EDAC.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1, or may be a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.
  • polynucleotide encoding a BDGI protein or polypeptide and "BDGI polypeptide coding sequence” are used interchangeably and may include a polynucleotide encoding BDGI or a polynucleoside further comprising additional coding and/or non-coding sequences. acid.
  • the present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the full-length human BDGI nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be used with commercially available cDNA libraries or as known to those skilled in the art.
  • the cDNA library prepared by the method was used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or BDGI protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant BDGI polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:
  • the invention also relates to agonists or inhibitors of BDGI polypeptides.
  • an inhibitor of a BDGI polypeptide refers to a substance that is capable of combating, inhibiting or reducing the activity or expression of a BDGI polypeptide.
  • Common inhibitors include antibodies, antisense nucleotides, interfering RNA (RNAi), certain natural extracts or compounds.
  • an agonist of a BDGI polypeptide refers to a substance that is capable of increasing, or promoting, the activity or expression of a BDGI polypeptide.
  • Common accelerators include BDGI fusion proteins, certain natural extracts or compounds, and the like.
  • an agonist or inhibitor of a BDGI polypeptide can be screened by an in vitro screening method.
  • the method includes:
  • control group is a culture system of a tumor cell expressing a BDGI polypeptide or a culture system of a tumor cell to which a BDGI polypeptide is added
  • test group is added with a test substance a culture system of a tumor cell expressing a BDGI polypeptide, or a culture system of a tumor cell to which a test substance and a BDGI polypeptide are added;
  • the increased migration ability of the tumor cells in the test group compared with the control group indicates that the test substance is an inhibitor of a potential BDGI polypeptide; and the decreased ability to migrate indicates that the test substance is a potential BDGI polypeptide.
  • Agent Pharmaceutical composition
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe therapeutically effective amount (e.g., 0.001 to 99% by weight) of a BDGI polypeptide of the present invention or a polynucleotide encoding the same (or an agonist thereof), and a pharmaceutically acceptable An acceptable carrier or excipient.
  • the human BDGI polypeptide is present in an amount of from 0.001 to 99.9% by weight, preferably from 0.01 to 95% by weight, more preferably from 0.1 to 90% by weight, most preferably from 0.5 to 80% by weight.
  • an effective amount refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given condition, routine experimentation can be used to determine the effective amount that the clinician can determine.
  • an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the invention.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration.
  • These vectors are well known to those of ordinary skill in the art.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the compositions of the present invention may contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • composition of the present invention may contain an additional anticancer drug in addition to the BDGI polypeptide as an active ingredient.
  • the additional anticancer drug is selected from the group consisting of: TNF-oc, TGF- ⁇ , IFN-oc, glycophosphoryl mustard, hematoporphyrin, lycopene betaine, Brucea javanica, and B. ⁇ (ie etoposide), dehydrated dulcitol, doxorubicin, tamoxifen, 5-fluorouracil, norcantharidin, difuran fluorouracil, cucurbitacin, harringtonine, oridonin, scorpion , Yunzhi glycopeptide, cytarabine, carbopol, paclitaxel, lentinan, flutamide, ifosfamide, umbrel, leuprolide acetate, deoxyfluorouridine, lopoplatin, Elinoxone, letrozole, teniposide, or a combination thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the pharmaceutical composition of the present invention can be administered orally, subcutaneously, intradermally, intravenously or the like.
  • the therapeutic dosage regimen can be a single dose regimen or a multiple dose regimen.
  • BDGI polypeptide When a pharmaceutical composition is used, a safe and effective amount of the BDGI polypeptide is administered to a mammal, especially a human. Specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • BDGI polypeptide of the present invention can be used alone to inhibit tumor cell migration, and can also be used in combination with other substances.
  • the BDGI polypeptide and its encoding polynucleotide as a pharmaceutical composition may be used alone or in combination with other polypeptides, polynucleotides, or in combination with other drugs and therapeutic means for the treatment of malignant tumors.
  • RNA from human bone marrow stromal cells was extracted using Trizol reagent (Life Technologies). Then, poly(A) mRNA is isolated from total RNA. After reverse transcription of poly (A) mRNA to form cDNA, the cDNA fragment was inserted into the vector by SuperScriptl l cloning kit (Life Technologies). At the cloning site, DH5a bacteria were transformed to form a cDNA plasmid library. The sequence of the 5' end of the randomly selected clone was determined by the dideoxy method. The determined cDNA sequence was compared with an existing public DNA sequence database, and it was found that the DNA sequence of one cDNA clone was a new full-length cDNA.
  • the DNA sequence contained in the new clone was bidirectionally determined by synthesizing a series of primers. Computer analysis indicated that the full-length cDNA contained in the clone was a new cDNA sequence (shown as SEQ ID NO: 1) encoding a new protein (as shown in SEQ ID NO: 2). This protein is the human growth inhibitory protein BDGI, and its coding gene is the human growth inhibitory protein BDGI gene.
  • Sequence SEQ ID NO: 1 is 1766 bp in length and includes a 146 bp 5' non-coding region and a 186 bp 3' non-coding region encoding a polypeptide of 477 amino acids. Theoretically, the molecular weight of the unglycosylated mature molecule is about 52 kD.
  • Example 2 Cloning of the coding sequence of human BDGI protein by RT-PCR
  • RNA in human bone marrow stromal cells was extracted with Trizol reagent, and 5 mg of total RNA was mixed with 1 mg of 01 igo-dT12-18 for reverse transcription.
  • the reverse transcription system was 20 ml, and 80 ml dd3 ⁇ 40 was added for dilution after the reaction.
  • the primers used for PCR amplification of BDGI are as follows:
  • the PCR reaction volume was 50 ml, which contained 10 ml of reverse transcription template, 0.5 mM primer, 0.2 mM dNTP, and 1 U rTaq DNA polymerase (Takara).
  • the amplification parameters were: 95 ° C, 15 sec, 58 ° C, 30 sec, 72 ° C, 90 sec.
  • the PCR product was subjected to 1.0% agarose gel electrophoresis.
  • the result of DNA sequence analysis revealed that the DNA sequence of the PCR product was identical to that of 147-1582 shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human BDGI
  • Northern blotting was performed as follows: The filter to be tested was placed in 10 ml of a pre-warmed hybridization solution at 68 ° C, pre-hybridized in a hybridization oven (Bellco) at 68 ° C for 30 minutes; the labeled cDNA probe was Denature at 95 ⁇ 100 °C for 2 ⁇ 5 minutes, add rapidly to the ice and add the hybridization solution (final concentration of cDNA probe is 2 ⁇ 10ng/ml or 1 ⁇ 2 X 106cpm/ml), mix well, at 68 °C Hybridize for 2 hours.
  • Bellco hybridization oven
  • the filter was rinsed several times with 2 'SSC, 0.05% SDS at room temperature, followed by shaking for 30 to 40 minutes, during which the lotion was changed several times. Subsequently, it was rinsed with 0. l 'SSC, 0.1% SDS at 50 ° C for 20 to 40 minutes. Finally, the filter was wrapped in a plastic wrap and exposed to -7 CTC for 24 to 48 hours.
  • Example 4 Prokaryotic expression of human BDGI protein
  • the full-length plasmid DNA of Example 1 was used as a template, and amplification was carried out using PCR oligonucleotide primers of the 5' and 3' ends of the sequence below to obtain human BDGI DNA as an insert.
  • the 5' oligonucleotide primer (upstream primer 2) sequence used in the PCR reaction is:
  • the 3' primer (upstream primer 2) sequence is:
  • the obtained PCR product was purified, digested with Nde I-Xhol, and then recombined with the expression vector plasmid pET-24a (+) (Novagen) according to a conventional method and transformed into competent E. coli DH5a, and cloned.
  • the positive clone was Nde.
  • l-Xhol was identified by enzyme digestion, and the product was analyzed by 0.8% agarose gel electrophoresis. After purification, it was purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE). It was confirmed by the sequence that the complete BDGI coding sequence has been inserted.
  • the positive DH5a clone expressing BDGI was inoculated into 100 ml of 2' YTA medium, cultured at 37 ° C, shaking at 300 rpm for 12 to 15 hours, and diluted 1:1 in pre-warmed 2 'YTA medium.
  • the human BDGI recombinant protein obtained in Example 4 was used to immunize an animal to produce an antibody, and the specific method is as follows.
  • the recombinant molecules are separated by chromatography and used. Separation can also be performed by SDS-PAGE gel electrophoresis, and the electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant.
  • Mice were intraperitoneally injected with 50-100 ⁇ g/0.2 ml of the emulsified protein. After 14 days, mice were intraperitoneally injected with a dose of 50-100 ⁇ g/0.2 ml with the same antigen emulsified with incomplete Freimd's adjuvant to boost the immunization.
  • Example 6 Construction of human BDGI eukaryotic expression vector and eukaryotic gene transfection
  • Example 2 using the full-length plasmid DNA of Example 1 as a template, amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human BDGI DNA as an insert.
  • the 5'-end oligonucleotide primer sequence (upstream primer 1) and the 3'-end primer (downstream primer 1) sequences used in the PCR reaction are the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the obtained PCR product was purified, digested with EcoR I-Hind III, and then recombined with the eukaryotic expression vector plasmid pcDNA3.1/myc-His (-) B (Invitrogen) according to a conventional method and transformed into competent Escherichia coli DH5a. Positive clones were picked and identified, purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE). It was confirmed by sequencing that the complete BDGI coding sequence has been inserted.
  • the BDGI eukaryotic expression plasmid DNA was transfected into human breast cancer MCF-7 cells and human lung cancer A549 cells by liposome LipofectAMINE reagent (Invitrogen), and the pcDNA3.1 plasmid vector was used as an unrelated control.
  • the main steps are: the plasmid DNA to be transfected is mixed with the liposome LipofectAMINE in a certain ratio, and allowed to react at room temperature for 45 minutes; 60 ⁇ 80% confluent cells grown in a 6-well cell culture plate are used, and serum is used in 0PTI-MEM.
  • MCF-7 cells and A549 cells transiently transfected with BDGI-expressing proteins in Example 6 were lysed with a cell lysate (Cell Signaling). Centrifuge at 4 ° C, 13, OOO rpm for 10 minutes. The supernatant was taken and protein quantification was performed using the BCA Protein Assay Kit (PIERCE). The protein sample was subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 100 V at 4 °C. Dye with Ponceau and mark the size and orientation. Block at room temperature for 2 hours (5% skim milk powder in TBST solution) and dilute the primary antibody with blocking solution and incubate for 1 hour at room temperature.
  • PIERCE BCA Protein Assay Kit
  • TBST (0. 05% Tween 20 in TBS solution) was washed for 15 minutes, 3 times, and the secondary antibody was diluted with blocking solution and incubated for 2 hours at room temperature.
  • the TBST was washed for 15 minutes, 3 times, and washed with TBS (10 mM Tri s-HC1, pH 8.0, 150 mM NaCl) for 15 minutes, then added with a chemiluminescent substrate (Pierce) for 1 minute, and rapidly sealed and auto-developed.
  • the primary antibody used for Western blot detection was the anti-BDGI antibody obtained in Example 5.
  • the secondary antibody is HRP-labeled anti-rabbit IgG (Cell Signaling).
  • the wells were inoculated into 96-well plates, and after 56 hours of culture, 3 H-TdR (1 Ci/well, Amersham) was added for further 16 hours, and then the cells were collected on a glass fiber filter paper using a multi-head cell harvester, followed by PBS. Trichloroacetic acid and ethanol were rinsed to remove the free 3 H-TdR, the filter paper was dried, and finally radioactively detected by a LS 6500 liquid scintillation counter, and the cpm value was counted.
  • the A549 cells overexpressing the BDGI protein in Example 6 in the logarithmic growth phase were seeded in a 96-well flat-bottomed plate, and each of the three wells was set and cultured at 37 ° C, 5% CO 2 .
  • 100 ml of medium containing 10% MTT (5 mg/ml, Sigma) was added, and incubation was continued for 4 hours.
  • the supernatant was carefully aspirated and 150 ml/well of DMS0 was added thereto, and incubated at 37 ° C for 10 minutes to dissolve the cells.
  • the 0D value of the 570 nm wavelength was measured by an enzyme-linked instrument.
  • the BDGI protein-expressing MCF-7 cells were prepared in a single cell suspension and counted under a microscope while taking 2 X DMEM medium (containing 20% FCS) and 0.6% low melting point agar. Serve, agar is heated and melted, cooled slightly, and then mixed. When cooled to about 37 °C, mix well with the prepared single cell suspension and add 6-well plate to 500 cells/well. Chromosomes with a diameter above ⁇ ⁇ were counted at 37 ° C, 5% CO 2 , for three weeks.
  • the MCF-7 cells overexpressing the BDGI protein in Example 6 in the logarithmic growth phase were inoculated into a 60 mm diameter petri dish at 5 ⁇ 10 5 cells/well, and when they reached 80% confluence, the blade was used. Draw a groove in the Petri dish as a marker, hold the blade, wipe the cells on one side of the blade with a sterile cotton ball, and immediately observe under the microscope to ensure that there is no cell residue on the side of the marked scratch line, and then use PBS. Wash 2 times, add fresh medium The culture was continued, and after 36 hours and 48 hours, the following two results were observed under the microscope: 1 number of cells migrating per field of view 2 maximum distance of cell migration per field.
  • Apoptosis was detected using the Annexin V/FITC kit (Bender), and the procedure was carried out according to the instructions, which are briefly described as follows: MCF-7 cells overexpressing BDGI protein in Example 6 were washed with PBS and then resuspended in Binding to the buffer. Dilute with Annexin V/FITC for 10 minutes at room temperature, PBS wash, resuspend in binding buffer, add PI, load immediately, analyze by flow cytometry, and analyze software as ModFit
  • the sufficiency of the 5% glutaraldehyde in the 5% glutaraldehyde is 0. 1M pity.
  • the cells were fixed at 4 ° C for 2 hours, and the cell pellet was washed twice with PBS in 0.1 M phosphate buffer (pH 7. 4) containing 1% citric acid. ), fixed at 4 ° C for 1 hour, washed twice with PBS, using gradient ethanol (50%, 70%,
  • the lmg BDGI polypeptide and the BDGI-His fusion protein prepared in Example 4 were separately subjected to a conventional mixing method.
  • Example 14 In vitro screening of BDGI inhibitors and agonists
  • Test substance 1 is the anti-BDGI antibody prepared in Example 5
  • Test substance 2 is the antisense sequence of the sequence of positions 147-1582 of SEQ ID NO: 1 (obtained according to a conventional molecular biological method)
  • the BDGI-His fusion protein prepared in Example 4 was an equal volume of physiological saline.
  • Test 1 and Test 2 increased the migration ability of MCF-7 cells compared to the control group, indicating that the two test substances were inhibitors of potential BDGI polypeptides; and Test 3 made MCF-7 The ability of the cells to migrate decreased, indicating that Test 3 is an agonist of the potential BDGI polypeptide.

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Abstract

La présente invention concerne des utilisations d'un inhibiteur de croissance dérivé des cellules stromales de la moelle osseuse humaine (BDGI) ou sa séquence codante pour la fabrication de compositions qui inhibent la migration de tumeurs et favorise l'apoptose des cellules tumorales. La protéine dans la présente invention est une protéine inhibitrice de la croissance qui inhibe la croissance et la migration de tumeurs et induit une apoptose des cellules tumorales.
PCT/CN2007/070626 2007-09-05 2007-09-05 Fonctions et utilisations d'un inhibiteur de croissance dérivé des cellules stromales de la moelle osseuse humaine Ceased WO2009030086A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1475503A (zh) * 2002-08-16 2004-02-18 浙江大学 新型人骨髓基质细胞来源的生长抑制因子，其编码序列及用途

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1475503A (zh) * 2002-08-16 2004-02-18 浙江大学 新型人骨髓基质细胞来源的生长抑制因子，其编码序列及用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FU, JINGXIN ET AL.: "Research in anti-tumor immune effective of a novel human bone marrow stromal cell -derived growth inhibitor (BDGI", CHINESE MASTER'S THESES FULL-TEXT DATABASE MEDICINE AND HEALTH SCIENCES, 15 August 2007 (2007-08-15), pages E072 - 367 *
TAO WANG ET AL.: "Bone Marrow Stromal Cell -derived Growth Inhibitor Inhibits Growth and Migration of Breast Cancer Cells via Induction of Cell Cycle Arres and Apoptosis", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 6, 2005, pages 4374 - 4382, XP055351581 *
WANG, TAO ET AL.: "Molecular Cloning and Characterization of a Novel Growth Inhibitor BDGI Associated with Breast Cancer", CHINESE DOCTORAL DISSERTATIONS & MASTER'S THESES FULL-TEXT DATABASE (MASTER) MEDICINE AND HEALTH SCIENCES, no. 3, 2004, pages E072 - 259 *

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