WO2023036300A1 - Dbc1调控细胞衰老及其应用 - Google Patents

Dbc1调控细胞衰老及其应用 Download PDF

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WO2023036300A1
WO2023036300A1 PCT/CN2022/118135 CN2022118135W WO2023036300A1 WO 2023036300 A1 WO2023036300 A1 WO 2023036300A1 CN 2022118135 W CN2022118135 W CN 2022118135W WO 2023036300 A1 WO2023036300 A1 WO 2023036300A1
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cells
dbc1
diseases
pharmaceutical composition
blm
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French (fr)
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李隽�
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中国医学科学院基础医学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to DBC1 regulating cell aging and its application.
  • Cell senescence refers to the shortening of telomeres caused by cell replication or external stress, which is manifested as the arrest of cell cycle.
  • Cell senescence is mainly presented in two forms. The first one refers to the shortening of telomeres after several times of cell replication, cell no longer proliferating, and cell growth stagnation. The second is that cellular senescence can be induced due to DNA damage or the activation of abnormal oncogenes.
  • senescent cells exhibit growth arrest, they remain metabolically active and are characterized by distinct molecular and cellular changes, including cell cycle arrest, morphological changes, activation of p53 and p16 senescence-related pathways, altered chromatin structure, and cytokine Significant changes in secreted factors such as chemokines and chemokines.
  • microcarriers to achieve three-dimensional cell culture that is, to prepare three-dimensional scaffolds or gels to provide a suitable growth environment for cells to achieve the effect of delaying cell aging.
  • Wei J et al. successfully maintained the activity of mouse embryonic stem cells using the preparation of three-dimensional hydrogel scaffolds (see Wei J, Han J, Zhao Y, et al. The importance of three-dimensional scaffold structure on stemness maintenance of mouse embryonicstem cells [J]. Biomaterials, 2014, 35(27): 7724-7733.).
  • the purpose of the present invention is to provide a drug for inhibiting cell senescence. Based on this purpose, the present invention adopts the following technical solutions:
  • the first aspect of the present invention provides an agent for preventing and/or treating aging-related diseases or disorders, the agent is an agent that promotes the interaction between DBC1 and BLM.
  • the agent is ML216.
  • the aging-related diseases or disorders are selected from lung diseases and disorders, and kidney diseases and disorders.
  • the pulmonary disease and condition is selected from the group consisting of pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and pulmonary diseases with age-related loss of lung function; for example, Pulmonary Fibrosis.
  • the renal diseases and conditions are selected from renal dysfunction, renal fibrosis and renal failure.
  • the second aspect of the present invention provides a pharmaceutical composition, which comprises a DBC1 promoter and the agent as described in the first aspect of the present invention.
  • the DBCl promoter is selected from a DBCl-containing expression vector and a DBCl protein.
  • the expression vector is a nucleic acid or a cell.
  • the cells are mammalian cells.
  • the mammalian cells are selected from lung cells and kidney cells.
  • the lung cells are lung fibroblasts; the kidney cells are human embryonic kidney cells.
  • the lung fibroblasts are human embryonic lung fibroblasts, such as human embryonic lung fibroblasts IMR-90.
  • the human embryonic kidney cells are selected from 293 cells, 293T cells and 293FT cells, such as 293T cells.
  • the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.
  • the dosage form of the pharmaceutical composition is tablet, capsule, granule, pill, drop pill, syrup, powder, powder, suppository, drop, emulsion, injection, solution or mixture Suspension.
  • the third aspect of the present invention provides a pharmaceutical composition for preventing and/or treating aging-related diseases or conditions, said pharmaceutical composition is as described in the second aspect of the present invention.
  • the aging-related disease or condition is as defined in the second aspect of the invention.
  • the fourth aspect of the present invention provides a method for preventing and/or treating aging-related diseases or conditions, the method comprising administering an effective amount of the agent as described in the first aspect of the present invention or the agent described in the first aspect of the present invention to a patient in need.
  • the second aspect provides the steps of the pharmaceutical composition.
  • the aging-related disease or condition is as defined in the second aspect of the invention.
  • the fifth aspect of the present invention provides a pharmaceutical composition, which includes a DBC1 accelerator.
  • the promoter includes an expression vector containing DBC1 or a recombinant protein of DBC1.
  • the sixth aspect of the present invention provides a reagent for promoting the interaction between DBC1 and BLM, and the reagent includes ML216.
  • the seventh aspect of the present invention provides a method for screening candidate drugs for the prevention or treatment of aging-related diseases or disorders, the method comprising the following steps:
  • the aging-related diseases or disorders include lung diseases and disorders, kidney diseases and disorders.
  • said pulmonary diseases and disorders include pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and pulmonary diseases with age-related loss of lung function, preferably Pulmonary Fibrosis.
  • said renal diseases and disorders include renal dysfunction, renal fibrosis, renal failure.
  • the eighth aspect of the present invention provides the purposes described in any one of the following:
  • cell senescence is cell senescence induced by DNA damage.
  • the cells are mammalian cells.
  • mammalian cells include lung cells and kidney cells.
  • lung cells are lung fibroblasts.
  • lung fibroblasts are human embryonic lung fibroblasts.
  • human embryonic lung fibroblasts are IMR-90.
  • kidney cells Further, the kidney cells.
  • kidney cells are human embryonic kidney cells.
  • human embryonic kidney cells include 293 cells, 293T cells, and 293FT cells.
  • human embryonic kidney cells are 293T cells.
  • the aging-related diseases or disorders include lung diseases and disorders, kidney diseases and disorders.
  • said pulmonary diseases and disorders include pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and pulmonary diseases with age-related loss of lung function, preferably Pulmonary Fibrosis.
  • said renal diseases and disorders include renal dysfunction, renal fibrosis, renal failure.
  • the medicine also includes pharmaceutically acceptable carriers and/or auxiliary materials.
  • the dosage form of the medicine includes tablet, capsule, granule, pill, drop pill, syrup, powder, powder, suppository, drop, emulsion, injection, solution or suspension.
  • the present invention provides the application of the accelerator of DBC1 in the preparation of medicines for preventing or treating aging-related diseases or diseases.
  • the present invention also provides the application of ML216 in the preparation of medicines for preventing or treating aging-related diseases or diseases.
  • the present invention also provides a method for screening candidate drugs for preventing or treating aging-related diseases or conditions.
  • Figure 1 is an experimental diagram proving that DBC1 knockdown promotes senescence and inhibits cell apoptosis in response to DNA damage, where A is the treatment of cisplatin (50 ⁇ M) with a living cell counting kit (Cell Counting Kit-8, CCK-8) The results of cell viability analysis of the DBC1 gene knockdown cells, the expression level of DBC1 gene in the DBC1 gene knockdown cells is shown in the right figure; B is the quantitative analysis using Annexin V staining and flow cytometry Apoptosis level experimental results of DBC1 gene knockdown cells treated with staurosporine (1 ⁇ M, 2 hours); C is Western Blot analysis of DBC1 gene knockdown cells treated with etoposide (50 ⁇ M, 24 hours) BCL-2, p53 and p21 protein levels experiment results; D is the cell cycle analysis results of DBC1 knockdown cells treated with etoposide (10 ⁇ M, 24 hours); E is the results of etoposide or DBC1 gene knock
  • FIG. 2 is an experimental diagram demonstrating that DBC1 regulates senescence induction by preserving BLM during DNA damage
  • A is the Western Blot analysis of BLM, p16 and p21 in DBC1 knockdown cells treated with etoposide (50 ⁇ M, 24 hours) The results of protein level experiments
  • B is the results of DBC1, BLM, p16 and p21 protein levels in BLM knockdown cells treated with etoposide (50 ⁇ M, 24 hours) by Western Blot
  • C is the results of BLM knockdown cells The results of cell cycle analysis
  • D is the result of SA- ⁇ -Gal staining analysis of BLM knockdown IMR-90 cells after etoposide-induced DIS
  • E is the result of etoposide (50 ⁇ M, 24 hours) Western Blot analysis of BLM and p21 protein levels in BLM knockdown cells overexpressing DBC1 after induction of DNA damage
  • F is SA- ⁇ of BLM gene knockdown IMR90 cells overexpressing DBC1
  • Figure 3 is an experimental diagram showing similar transcriptional patterns associated with aging between BLM knockout cells and DBC1 knockout cells by RNA sequencing analysis, where A is the dimension of common differentially expressed genes in DBC1 knockout cells and BLM knockout cells En diagram; B is the result of Gene Ontology analysis of common differentially expressed genes in DBC1 gene knockdown cells and BLM gene knockdown cells; C is the KEGG enrichment analysis of DBC1 gene knockdown cells and BLM gene knockdown cells D is the experimental result diagram of the effect of etoposide treatment (50 ⁇ M, 24 hours) on the expression levels of senescence-related genes in DBC1 knockout cells and BLm knockout cells.
  • A is the dimension of common differentially expressed genes in DBC1 knockout cells and BLM knockout cells En diagram
  • B is the result of Gene Ontology analysis of common differentially expressed genes in DBC1 gene knockdown cells and BLM gene knockdown cells
  • C is the KEGG enrichment analysis of DBC1 gene knockdown cells and BLM gene knockdown
  • FIG. 4 is an experimental diagram demonstrating that DBC1 prevents the degradation of BLM and inhibits the induction of p21 after DNA damage by binding to it.
  • A is treated with etoposide (100 ⁇ M) or Z-VAD(OMe)-FMK for 24 hours by Western Blot analysis
  • B is the experimental results of the interaction between DBC1 and BLM detected by co-immunoprecipitation experiments
  • C is the results of co-immunoprecipitation experiments of full-length or truncated DBC1 and BLM
  • bottom panel is a schematic structure diagram of DBC1 full-length and truncated mutants (FL represents full-length, ⁇ Nudix represents the deletion of the Nudix domain (residues 339-463), ⁇ NT represents the N-terminal domain (residues 1-263) Deletion);
  • D is the experimental results of BLM and p21 protein levels in DBC
  • Figure 5 is an experimental diagram proving that ML216 protects BLM from degradation and inhibits DNA damage-induced senescence by promoting DBC1-BLM interaction
  • A is the experimental result diagram of the effect of ML216 on DBC1-BLM interaction detected by co-immunoprecipitation experiment
  • B is the experimental results of BLM and p21 protein levels analyzed by Western Blot after treating DBC1 gene knockdown cells with etoposide (100 ⁇ M) or ML216 (50 ⁇ M) for 24 hours
  • C is the results of DBC1 and BLM under different concentrations of ML216
  • D is the results of Western Blot detection of BLM, ⁇ H2AX and p21 protein levels in 293T cells treated with different concentrations of ML216 (24 hours)
  • E is the results of experiments with etoposide or etoposide with different concentrations ML216 co-treatment to induce the number of SA- ⁇ -Gal positive cells in IMR90
  • Figure 6 is an experimental diagram proving that ML216 can reduce fibrosis and improve lung function in IPF mice, wherein, A is a schematic diagram of the experimental design of bleomycin-induced pulmonary fibrosis in C57BL/6J mice; B is the lung function of mice Statistical diagram of parameter measurement; C is a representative diagram of Masson staining in mouse lung; D is the experimental result diagram of measuring mouse lung tissue collagen content by hydroxyproline assay; E is the fibrosis marker gene COL1A1, MMP2, Statistical graph of mRNA expression levels of FN1, TGF- ⁇ , and CTGF in mouse lung.
  • Figure 7 is an experimental diagram proving that ML216 can delay cell aging at the molecular level in the lungs of IPF mice, wherein, A is a statistical diagram of the protein levels of SASP factors (IL-6, MCP-1, CXCL-1) in the lungs detected by ELISA; B is the statistical chart of the protein level of SASP factors (IL-6, MCP-1, CXCL-1) in the serum detected by ELISA; C is the experimental result chart and statistical analysis chart of the p21 protein level in mouse lung analyzed by Western Blot; D Is a representative image of the aging level of mouse lung analyzed by SA- ⁇ -Gal staining; E is a quantitative statistical map of SA- ⁇ -Gal positive cells in mouse lung.
  • A is a statistical diagram of the protein levels of SASP factors (IL-6, MCP-1, CXCL-1) in the lungs detected by ELISA
  • B is the statistical chart of the protein level of SASP factors (IL-6, MCP-1, CXCL-1) in the serum detected by
  • Figure 8 is an experimental diagram for detecting the effect of DBC1 overexpression on cell survival rate and apoptosis level, wherein, A is the result after treatment with cisplatin (50 ⁇ M) using a living cell counting kit (Cell Counting Kit-8, CCK-8) The results of the cell viability analysis experiment of cells overexpressing the DBC1 gene, the overexpression level of DBC1 is shown in the right figure; B is quantitative analysis by annexin V staining and flow cytometry after staurosporine (1 ⁇ M, 2 hours) Representative scatter plots and statistical charts for quantitative analysis of apoptosis levels of DBC1 overexpressed cells.
  • the invention provides a pharmaceutical composition, which includes a DBC1 accelerator.
  • the “DBC1” refers to the gene whose Gene ID is 57805.
  • the promoter of DBC1 refers to any substance that can improve the stability of DBC1 gene or expression product, upregulate the expression of DBC1, increase the effective time of DBC1 or promote the transcription of DBC1 gene, these substances can be used in the present invention, as for Substances useful for up-regulating the expression of the DBC1 gene, thereby being useful for preventing or treating aging-related diseases or conditions.
  • the promoter includes an expression vector containing DBC1 or a recombinant protein of DBC1.
  • the promoter of DBC1 is an expression vector containing DBC1.
  • the expression vector usually also contains a promoter, an origin of replication and/or a marker gene and the like.
  • Said expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as karithromycin, gentamicin, hygromycin, ampicillin resistance genes .
  • the expression vectors are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses and the like.
  • the introduction of expression vectors into host cells can be performed by electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, virus infection, lipofection, and binding to cell membrane-permeable peptides. and other well-known methods.
  • a senescence-associated disease or disorder may also be referred to herein as a senescent cell-associated disease or disorder.
  • Aging-associated diseases or disorders include, for example, cardiovascular diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, pulmonary diseases and disorders, eye diseases and disorders, metabolic diseases and disorders, neurological diseases and disorders (e.g., neurological degenerative diseases and disorders); age-related diseases and disorders caused by aging; age-related diseases; skin diseases and disorders; and transplantation-related diseases and disorders.
  • the prominent feature of aging is the gradual loss or degradation of function that occurs at the molecular, cellular, tissue, and biological levels.
  • Age-related degeneration causes well-recognized pathologies such as muscular atrophy, atherosclerosis and heart failure, osteoporosis, pulmonary insufficiency, renal failure, neurodegeneration (including macular degeneration, Alzheimer's disease, and Parkinson's disease) and many other diseases.
  • said aging-related diseases or disorders are pulmonary diseases and disorders, including but not limited to pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis , a lung disease with age-related loss of lung function, preferably pulmonary fibrosis.
  • pulmonary diseases and disorders including but not limited to pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis , a lung disease with age-related loss of lung function, preferably pulmonary fibrosis.
  • the aging-related diseases or disorders are renal diseases and disorders, and the renal diseases and disorders include renal dysfunction, renal fibrosis, and renal failure.
  • the medicament for treating aging-related diseases or diseases of the present invention also includes pharmaceutically acceptable carriers and/or adjuvants.
  • pharmaceutically acceptable carrier includes any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening agents or emulsifying agents suitable for preparing the particular dosage form desired. Agents, preservatives, solid adhesives, lubricants, etc.
  • materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethylcellulose Sodium veg, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil , corn oil, and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline ; Ringer's solution; ethanol and phosphate buffered saline, and, and,
  • various methods well known in the art can be used to administer the medicament for preventing or treating aging-related diseases or disorders to mammals. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, sustained release administration, etc.
  • the effective amount of the accelerator of DBC1 or ML216 (CAS No.: 1430213-30-1) of the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials).
  • the factors include but are not limited to: the accelerator of DBC1 or the pharmacokinetic parameters of ML216 such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, and the patient's immunity condition, route of administration, etc. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.
  • cell includes both prokaryotic and eukaryotic cells.
  • prokaryotic cells include Escherichia coli, Bacillus subtilis, and the like.
  • eukaryotic cells include yeast cells, insect cells, and mammalian cells. Preferably, the cells are mammalian cells.
  • the mammalian cells are lung cells, preferably lung fibroblasts, in a specific embodiment of the present invention, the lung fibroblasts are human embryonic lung fibroblasts, and the human Embryonic lung fibroblasts include but not limited to WI-38, KMB17, IMR-90, MRC-5, 2BS, preferably IMR-90.
  • the mammalian cells are kidney cells, and the kidney cells include but are not limited to monkey kidney CVI cell lines (such as COS-7, ATCC CRL1651); human embryonic kidney lines (such as 293, 293T, 293FT); young hamster kidney cells (such as BHK, ATCC CCL10); monkey kidney cells (such as CVI ATCC CCL70); vero cells (such as VERO-76, ATCC CRL-1587); canine kidney cells (such as MOCK, ATCC CCL34) etc.
  • monkey kidney CVI cell lines such as COS-7, ATCC CRL1651
  • human embryonic kidney lines such as 293, 293T, 293FT
  • young hamster kidney cells such as BHK, ATCC CCL10
  • monkey kidney cells such as CVI ATCC CCL70
  • vero cells such as VERO-76, ATCC CRL-1587
  • canine kidney cells such as MOCK, ATCC CCL34 etc.
  • the present invention provides a method for screening candidate drugs for preventing or treating aging-related diseases or conditions, the method comprising the following steps:
  • the system expressing or containing DBC1 may be a cell (or cell culture) system, and the cells may be endogenously expressing DBC1 cells; or may be recombinantly expressing DBC1 cells.
  • the system expressing or containing DBC1 can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, a rabbit, a sheep, a monkey, etc. )wait.
  • treatment generally refers to the treatment of humans or animals (e.g., as applied by veterinary medicine), wherein some desired therapeutic effect is achieved, e.g., inhibiting the development of a condition (including slowing the rate of development, arresting the development), Improve disease and cure disease. Also included is treatment as a prophylactic measure (eg, prophylaxis). Also included in the term “treatment” is the use of a patient who has not yet developed a condition, but is at risk of developing it.
  • DBC1 knockdown 293T cells pRS-DBC1, Origene, TR303704
  • DBC1 overexpression 293T cells pMSCV-Flag-DBC1, self-built plasmid
  • annexin V staining FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, 556547
  • flow cytometry was used to quantitatively analyze Staurosporum Apoptotic levels of DBC1 knockdown cells or DBC1 overexpression 293T cells treated with DBC1 (1 ⁇ M, 2 hours).
  • Western Blot was used to detect the expression level of DBC1 gene in DBC1 knockdown cells or DBC1 overexpression 293T cells.
  • DNA damage induced senescence DNA damage induced senescence
  • SA- ⁇ -Gal staining analysis was performed on DBC1 gene knockdown IMR-90 cells induced by etoposide to senescence, and the effect of DBC1 gene knockdown on IMR-90 cell senescence was studied.
  • the cells treated with DMSO were the control group .
  • the method of DIS induction was as follows: the IMR-90 cells of early passage were treated with etoposide (20 ⁇ M) for 24 hours at the 16th passage.
  • the medium was replaced with fresh medium to replace the cells, and continued to grow for 7 days, and the fresh medium was frequently replaced within 7 days to make the IMR-90 cells senescent.
  • SA- ⁇ -Gal staining cells were fixed in G/F fixative (50% glutaraldehyde and 37% formaldehyde in PBS) for 5 minutes at room temperature.
  • RT-PCR analysis to detect the expression levels of IL-6, IL-8, IL-1 ⁇ , CXCL-1, CXCL-2, MMP-2, MMP-3 and MMP-9 in DBC1 knockdown IMR-90 cells .
  • Ki-67 staining was performed to analyze the proliferation of DBC1 gene knockdown IMR-90 cells, wherein the cells treated with DMSO were the control group.
  • knocking down the DBC1 gene can increase the cell survival rate after DNA damage
  • the overexpression of DBC1 can increase cell death rate in response to DNA damage, especially increase the level of apoptosis.
  • p21 is a CDK inhibitor that prevents cell cycle progression through the G1/S phase, as shown in Figure 1D, DBC1 knockdown cells exhibit prolonged G1 after DNA damage as p21 induction levels increase Period block.
  • SA- ⁇ -gal senescence-associated galactosidase staining positive cells
  • SASP senescence-associated secretory phenotype
  • RNA sequencing analysis was performed on DBC1 knockdown cells or BLM knockdown 293T cells, respectively.
  • the common differentially expressed genes in DBC1 knockdown cells and BLM knockdown 293T cells are presented in the form of a Venn diagram, and Gene Ontology analysis is performed on these differentially expressed genes, -log10(P value ) as a function of classification, satisfying a P value ⁇ 0.05.
  • the common differentially expressed genes of DBC1 knockdown cells and BLM knockdown cells compared with control cells.
  • DBC1 was transfected into BLM-deficient cells, and after DNA damage was induced, as more DBC1 existed, more BLM protein was retained, and the level of p21 also decreased. .
  • transfection of DBC1 into BLM-deficient IMR-90 cells can reduce the number of SA- ⁇ -gal positive cells and inhibit the level of senescence (as shown in F of Figure 2).
  • knockdown of BLM had no effect on the expression level of BCL-2 or the level of apoptosis. Influence.
  • RNA sequencing was performed on DBC1 knockdown cells and BLM knockdown cells, and the transcripts that were commonly changed in the two sets of data were analyzed to obtain gene expression profiles (GEPs) with 200 gene overlaps.
  • GEPs gene expression profiles
  • GO enrichment analysis in Biological Process showed that "Cellular Aging" was one of the most enriched up-regulated pathways common to both datasets (shown in Figure 3B).
  • KEGG analysis of genes co-regulated by DBC1 and BLM revealed that the most enriched pathways were related to oxidative phosphorylation, Huntington's disease, metabolic pathways, Parkinson's disease, and Alzheimer's disease, all of which are aging-related diseases if Defects in function lead to senescence (as shown in Figure 3, C).
  • D which provides evidence for functional overlap between BLM and DBC1 in regulating cellular senescence.
  • Example 3 DBC1 protects BLM from degradation by combining with BLM
  • the present invention uses the pan-caspase inhibitor Z-VAD(OMe)-FMK to treat the cells.
  • Western Blot analysis was performed on BLM and p21 protein levels in DBC1 knockdown 293T cells treated with etoposide (100 ⁇ M) or Z-VAD(OMe)-FMK for 24 hours.
  • Z-VAD(OME)-FMK can reduce the loss of BLM after DNA damage to a level comparable to that of the control group, and the level of p21 is also reduced accordingly. This result demonstrates that the presence of DBC1 reduces the cleavage of BLM by DNA damage-activated caspases.
  • Example 4 ML216 protects BLM from degradation and inhibits DNA damage-induced senescence by promoting DBC1-BLM interaction.
  • DBC1 knockdown 293T cells were treated with etoposide (100 ⁇ M) or ML216 (50 ⁇ M) for 24 hours, and then BLM and p21 protein levels were analyzed by Western Blot.
  • the number of SA- ⁇ -Gal positive cells was detected after the DIS induction of IMR90 cells was induced by etoposide or co-treatment with etoposide and different concentrations of ML216.
  • RT-PCR was used to detect p16, p21, IL-1 ⁇ , Expression levels of IL-6 and CXCL-1.
  • HR homologous recombination
  • the HR procedure is as follows: pcDNA3.1-HR plasmid (self-built plasmid), pCBASceI plasmid (Addgene, 26477) and dsRed2-N1 plasmid (Addgene, 54493) were transfected into cells. Drug treatment was performed 48 hours after transfection, and final cells were quantified by flow cytometry. The ratio of GFP-positive cells to RFP-positive cells was used to calculate HR repair efficiency.
  • ML216 significantly reduces the positive senescence of SA- ⁇ -Gal in cells induced by etoposide in DIS cell ratio. As shown in Figure 5G, ML216 also reduced the expression levels of SASP factors IL-1a, IL-6 and CXCL1.
  • Example 5 ML216 reduces aging-related pathological changes in idiopathic pulmonary fibrosis (IPF) mouse model
  • mice were injected intraperitoneally with ML216 (0.5 mg/kg) or vehicle, twice a week for 3 weeks in total. After 3 weeks, the lung function parameters of the mice were detected, including: dynamic compliance, peak compliance and string compliance.
  • Masson staining Masson trichrome staining Kit, Solarbio, G1343
  • H&E staining was performed by the Department of Histology, State Key Laboratory of Medical Molecular Biology, Peking Union Medical College Medical College
  • the collagen content of mouse lung tissue was determined by hydroxyproline (Hydroxyproline Assay Kit, Solarbio, BC0255).
  • mice CXCL1/KC(IL-8) ELISA Kit Absin, ABS520017
  • Mouse CCL2/JE/MCP-1 ELISA Kit Absin, ABS520016
  • Mouse IL-6 ELISA Kit Absin, ABS520004
  • SA- ⁇ -Gal staining For tissue SA- ⁇ -Gal staining, cryosections were fixed in ice-cold fixative (cold PBS containing 2% formaldehyde and 0.2% glutaraldehyde) for 7 minutes.
  • ML216 administration significantly reduced the protein levels of IL-6 in the lung and MCP-1 in the serum, while other factors showed a downward trend (as shown in Figure 7A and Figure 7B).
  • the induction rate of p21 in the lungs of the ML216 treatment group was significantly lower than that of the control group (as shown in C of FIG. 7 ).

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Abstract

DBC1调控细胞衰老及其应用。DBC1促进剂在制备预防或治疗衰老相关疾病或病症的药物中的应用。ML216在制备预防或治疗衰老相关疾病或病症的药物中的应用。为预防或治疗衰老相关疾病或病症提供了新途径。

Description

DBC1调控细胞衰老及其应用
本申请要求申请日为2021/9/10的中国专利申请2021110591012的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体涉及DBC1调控细胞衰老及其应用。
背景技术
细胞衰老是指细胞复制或者外界应力导致的端粒缩短，表现为细胞周期的停滞。细胞衰老主要以两种形式呈现，第一种是指细胞经过数次复制后，端粒缩短，细胞不再增殖，出现细胞生长停滞状态。第二种是由于DNA损伤或者是异常癌基因的激活可以诱导细胞衰老。尽管衰老的细胞表现为生长停滞，他们依然具有代谢活性，并且具有明显的分子和细胞变化特征，包括细胞周期停滞、形态学变化、p53和p16衰老相关通路的激活、染色质结构改变以及细胞因子和趋化因子等分泌因子的显著变化。
虽然衰老是不可避免和不可逆转的，但衰老的过程和速度可被人为干预。常见的细胞衰老干预方法包括：
1.制备低氧状态或添加活性分子、多酚药物分子来延缓细胞衰老。例如，Eom等利用FGF-2、FGF-4、EGF、HGF生长因子来维持骨髓间充质干细胞的干性(参见Young,Woo,Eom,et al.The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells[J].Biochemical and Biophysical ResearchCommunications,2014,445(1):16-22.)。Li Peng Cheng等利用低氧环境来增强胶质母细胞瘤癌症干细胞的干性(参见Li P C,Zhou C,Xu L,et al.Hypoxia enhances stemnessof cancer stem cells in glioblastoma:An in vitro study[J].InternationalJournal of Medical Sciences,2013,10(4):399-407.)。
2.利用微载体实现细胞三维培养，即制备三维支架或凝胶为细胞提供适宜的生长环境，来达到延缓细胞衰老的作用。例如Wei J等利用三维水凝胶支架的制备，成功维持了小鼠胚胎干细胞的活性(参见Wei J,Han J,Zhao Y,et al.The importance of three-dimensional scaffold structure on stemness maintenance of mouse embryonicstem cells[J].Biomaterials,2014,35(27):7724-7733.)。
然而这些方法存在一些缺陷，如：活性分子的直接添加往往药物可控性较差，而三 维培养方法存在收集效率低等问题。因此，本领域仍然需要寻找新的抑制细胞衰老的药物。
发明内容
鉴于现有技术的不足，本发明的目的在于提供一种抑制细胞衰老的药物，基于该目的，本发明采用了如下技术方案：
本发明第一方面提供了一种用于预防和/或治疗衰老相关疾病或病症的试剂，所述试剂为促进DBC1与BLM相互作用的试剂。
在一些实施方案中，所述试剂为ML216。
本发明中，所述衰老相关疾病或病症选自肺部疾病和病症，以及肾脏疾病和病症。
在一些实施方案中，所述肺部疾病和病症选自肺纤维化、慢性阻塞性肺疾病、哮喘、囊性纤维化、肺气肿、支气管扩张和年龄相关肺功能丧失的肺疾病；例如为肺纤维化。
所述肾脏疾病和病症选自肾功能障碍、肾纤维化和肾衰竭。
本发明第二方面提供了一种药物组合物，所述药物组合物包括DBC1促进剂和如本发明第一方面所述的试剂。
在一些实施方案中，所述DBC1促进剂选自含有DBC1的表达载体和DBC1蛋白。
在一些实施方案中，所述表达载体为核酸或细胞。
在一些实施方案中，所述细胞为哺乳动物细胞。
在一些较佳实施方案中，所述哺乳动物细胞选自肺细胞和肾脏细胞。
在一些实施方案中，所述的肺细胞为肺成纤维细胞；所述肾脏细胞为人胚肾细胞。
在一些具体实施方案中，所述的肺成纤维细胞为人胚肺成纤维细胞，例如为人胚肺成纤维细胞IMR-90。
在一些具体实施方案中，所述人胚肾细胞选自293细胞、293T细胞和293FT细胞，例如为293T细胞。
在一些实施方案中，所述药物组合物还包括药学上可接受的载体和/或辅料。
在一些较佳实施方案中，所述药物组合物的剂型为片剂、胶囊剂、颗粒剂、丸剂、滴丸剂、糖浆剂、粉剂、散剂、栓剂、滴剂、乳剂、注射液、溶液或混悬液。
本发明第三方面提供了一种用于预防和/或治疗衰老相关疾病或病症的药物组合物，所述药物组合物如本发明第二方面所述。
在一些实施方案中，所述衰老相关疾病或病症如本发明第二方面所定义。
本发明第四方面提供了一种预防和/或治疗衰老相关疾病或病症的方法，所述方法包 括向有需要的患者施用有效量的如本发明第一方面所述的试剂或者如本发明第二方面提供了所述的药物组合物的步骤。
在一些实施方案中，所述衰老相关疾病或病症如本发明第二方面所定义。
本发明第五方面提供了一种药物组合物，所述的药物组合物包括DBC1的促进剂。
进一步，所述促进剂包括含有DBC1的表达载体或者DBC1的重组蛋白。
本发明第六方面提供了一种促进DBC1与BLM相互作用的试剂，所述的试剂包括ML216。
本发明第七方面提供了一种筛选预防或治疗衰老相关疾病或病症的候选药物的方法，所述的方法包括以下步骤：
(1)用待测试物质处理表达或含有DBC1的体系；
(2)检测所述体系中DBC1的表达水平；
(3)选择可上调DBC1表达水平的物质为候选药物。
进一步，所述的衰老相关疾病或病症包括肺疾病和病症、肾脏疾病和病症。
作为一种实施方案，所述的肺疾病和病症包括肺纤维化、慢性阻塞性肺疾病、哮喘、囊性纤维化、肺气肿、支气管扩张、和年龄相关肺功能丧失的肺疾病，优选为肺纤维化。
作为一种实施方案，所述的肾脏疾病和病症包括肾功能障碍、肾纤维化、肾衰竭。
本发明第八方面提供了如下任一项所述的用途：
(1)本发明第一方面所述的药物组合物或本发明第二方面所述的试剂在制备抑制细胞衰老的药物中的用途；
(2)本发明第一方面所述的药物组合物或本发明第二方面所述的试剂在制备预防或治疗衰老相关疾病或病症的药物中的用途；
(3)DBC1在筛选预防或治疗衰老相关疾病或病症的候选药物中的用途。
进一步，所述的细胞衰老为DNA损伤诱导的细胞衰老。
进一步，所述的细胞为哺乳动物细胞。
进一步，所述的哺乳动物细胞包括肺细胞、肾脏细胞。
进一步，所述的肺细胞为肺成纤维细胞。
进一步，所述的肺成纤维细胞为人胚肺成纤维细胞。
进一步，所述的人胚肺成纤维细胞为IMR-90。
进一步，所述的肾脏细胞。
进一步，所述的肾脏细胞为人胚肾细胞。
进一步，所述的人胚肾细胞包括293细胞、293T细胞、293FT细胞。
进一步，所述的人胚肾细胞为293T细胞。
进一步，所述的衰老相关疾病或病症包括肺疾病和病症、肾脏疾病和病症。
作为一种实施方案，所述的肺疾病和病症包括肺纤维化、慢性阻塞性肺疾病、哮喘、囊性纤维化、肺气肿、支气管扩张、和年龄相关肺功能丧失的肺疾病，优选为肺纤维化。
作为一种实施方案，所述的肾脏疾病和病症包括肾功能障碍、肾纤维化、肾衰竭。
进一步，所述的药物还包括药学上可接受的载体和/或辅料。
进一步，所述药物的剂型包括片剂、胶囊剂、颗粒剂、丸剂、滴丸剂、糖浆剂、粉剂、散剂、栓剂、滴剂、乳剂、注射液、溶液或混悬液。
本发明的优先和有益效果：
本发明提供了DBC1的促进剂在制备预防或治疗衰老相关疾病或病症的药物中的应用。
本发明还提供了ML216在制备预防或治疗衰老相关疾病或病症的药物中的应用。
本发明还提供了一种筛选预防或治疗衰老相关疾病或病症的候选药物的方法。
附图说明
图1为证明DBC1敲低促进衰老、抑制细胞凋亡以应对DNA损伤的实验图，其中，A是使用活细胞计数试剂盒(Cell Counting Kit-8，CCK-8)对顺铂(50μM)处理后的DBC1基因敲低细胞进行细胞存活率分析实验结果图，DBC1基因在DBC1基因敲低细胞中的表达水平如其右图所示；B是使用膜联蛋白V染色法和流式细胞术定量分析经星形孢菌素(1μM，2小时)处理的DBC1基因敲低细胞的凋亡水平实验结果图；C是采用Western Blot分析经依托泊苷(50μM，24小时)处理的DBC1基因敲低细胞中BCL-2、p53和p21蛋白水平实验结果图；D是对经依托泊苷(10μM，24小时)处理的DBC1基因敲低细胞进行细胞周期分析实验结果图；E是对经依托泊苷或二甲基亚砜(对照)诱导的DBC1基因敲低IMR-90细胞进行SA-β-Gal染色分析实验结果图及统计分析图；F是采用RT-PCR分析检测DBC1基因敲低细胞中IL-6、IL-8、IL-1α、CXCL-1、CXCL-2、MMP-2、MMP-3和MMP-9的蛋白水平统计图；G是在依托泊苷诱导细胞DNA损伤后进行Ki-67染色分析DBC1基因敲低细胞的增殖情况实验结果图及统计分析图。
图2为证明DBC1通过在DNA损伤期间保存BLM来调节衰老诱导的实验图，其中A是采用Western Blot分析经依托泊苷(50μM，24小时)处理的DBC1基因敲低细胞中BLM、p16和p21蛋白水平实验结果图；B是采用Western Blot分析经依托泊苷(50μM，24小时)处理的BLM基因敲低细胞中DBC1、BLM、p16和p21蛋白水平实验结果图；C是BLM敲低细胞的细胞周期分析结果图；D是经依托泊苷诱导DIS后对BLM敲低 IMR-90细胞进行SA-β-Gal染色分析实验结果图及统计图；E是经依托泊苷(50μM，24小时)诱导DNA损伤后采用Western Blot分析过表达DBC1的BLM敲低细胞中BLM和p21蛋白水平实验结果图；F是经依托泊苷诱导DIS后对过表达DBC1的BLM基因敲低IMR90细胞进行SA-β-Gal染色分析实验结果图及统计分析图。
图3为RNA测序分析显示BLM基因敲除细胞和DBC1基因敲除细胞之间与衰老相关的转录模式相似的实验图，其中A是DBC1敲低细胞和BLM敲低细胞中共同差异表达基因的维恩图；B是对DBC1基因敲低细胞和BLM基因敲低细胞中常见的差异表达基因进行基因本体论分析实验结果图；C是运用KEGG富集分析DBC1基因敲低细胞和BLM基因敲低细胞的共同差异表达基因实验结果图；D是分析依托泊苷处理(50μM，24小时)对DBC1基因敲除细胞和BLm基因敲除细胞衰老相关基因表达水平的影响实验结果图。
图4为证明DBC1通过与其结合阻止BLM降解并抑制DNA损伤后p21的诱导的实验图，其中，A是采用Western Blot分析经依托泊苷(100μM)或Z-VAD(OMe)-FMK处理24小时的DBC1基因敲低细胞中的BLM和p21蛋白水平实验结果图，B是采用免疫共沉淀实验检测DBC1与BLM相互作用实验结果图，C是将全长或截断DBC1与BLM进行免疫共沉淀实验结果图，底部图为DBC1全长和截断突变体的示意结构图(FL代表全长，δNudix代表Nudix结构域的缺失(残基339-463)，δNT代表N末端结构域(残基1-263)的缺失)；D是经依托泊苷诱导(50μM，24小时)细胞DNA损伤后通过Western Blot分析DBC1敲低细胞中的BLM和p21蛋白水平实验结果图。
图5为证明ML216通过促进DBC1-BLM相互作用保护BLM免于降解并抑制DNA损伤诱导的衰老的实验图，其中，A是采用免疫共沉淀实验检测ML216对DBC1-BLM相互作用的影响实验结果图，B是用依托泊苷(100μM)或ML216(50μM)处理DBC1基因敲低细胞24小时后采用Western Blot分析BLM和p21蛋白水平实验结果图，C是在不同浓度的ML216下进行DBC1与BLM的体外免疫沉淀实验结果图，D是采用Western Blot检测经不同浓度ML216处理(24小时)的293T细胞的BLM、γH2AX和p21蛋白水平实验结果图，E是用依托泊苷或依托泊苷与不同浓度的ML216共处理诱导IMR90细胞DIS后检测SA-β-Gal阳性细胞数的实验图；F是用依托泊苷或依托泊苷与不同浓度的ML216共处理诱导IMR90细胞DIS后检测SA-β-Gal阳性细胞数的统计图；G是采用RT-PCR检测诱导DIS后IMR90细胞中p16、p21、IL-1α、IL-6、CXCL-1的表达水平统计图；H是采用不同浓度ML216处理293T细胞24小时后检测其同源重组(HR)修复效率实验结果图；I是检测经ML216(50μM，24小时)处理的DBC1基因敲除293T细胞 的HR修复效率实验结果图。
图6为证明ML216可降低IPF小鼠的纤维化和改善肺功能的实验图，其中，A是博莱霉素诱导C57BL/6J小鼠肺纤维化的实验设计示意图；B是小鼠的肺功能参数测量统计图；C是小鼠肺部Masson染色的代表性图；D是采用羟脯氨酸测定法测定小鼠肺组织胶原蛋白含量实验结果图；E是纤维化标志物基因COL1A1、MMP2、FN1、TGF-β、CTGF在小鼠肺中的mRNA表达水平统计图。
图7为证明ML216可以延缓IPF小鼠肺部分子水平细胞衰老的实验图，其中，A是采用ELISA检测肺中SASP因子(IL-6、MCP-1、CXCL-1)的蛋白水平统计图；B是采用ELISA检测血清中SASP因子(IL-6、MCP-1、CXCL-1)的蛋白水平统计图；C是采用Western Blot分析小鼠肺中p21蛋白水平实验结果图及统计分析图；D是采用SA-β-Gal染色分析小鼠肺部衰老水平的代表性图像；E是小鼠肺SA-β-Gal阳性细胞的定量统计图。
图8是检测DBC1过表达对细胞存活率、凋亡水平的影响实验图，其中，A是使用活细胞计数试剂盒(Cell Counting Kit-8，CCK-8)对顺铂(50μM)处理后的过表达DBC1基因的细胞进行细胞存活率分析实验结果图，DBC1过表达水平如其右图所示；B是使用膜联蛋白V染色法和流式细胞术定量分析经星形孢菌素(1μM，2小时)处理的DBC1过表达细胞凋亡水平的代表性散点图和定量分析统计图。
具体实施方式
DBC1的促进剂
本发明提供了一种药物组合物，所述的药物组合物包括DBC1的促进剂。
所述的“DBC1”是指Gene ID为57805的基因。
所述的DBC1的促进剂是指任何可提高DBC1基因或表达产物稳定性、上调DBC1的表达、增加DBC1的有效作用时间或促进DBC1基因的转录的物质，这些物质均可用于本发明，作为对于上调DBC1基因的表达有用的物质，从而可用于预防或治疗衰老相关疾病或病症。
所述的促进剂包括含有DBC1的表达载体或者DBC1的重组蛋白。作为本发明的一种优选方式，所述的DBC1的促进剂为一种含有DBC1的表达载体。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。
本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个 或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性基因。
在本发明中，所述表达载体是本领域已知的各种载体，如市售的载体、包括质粒、粘粒、噬菌体、病毒等。表达载体向宿主细胞中的导入可以使用电穿孔法、磷酸钙法、脂质体法、DEAE葡聚糖法、显微注射、病毒感染、脂质体转染、与细胞膜透过性肽的结合等周知的方法。
衰老相关疾病或病症
本发明提供了用于治疗衰老相关疾病或病症的药物。衰老相关疾病或病症在本文还可以被称为衰老细胞相关疾病或病症。衰老相关疾病或病症包括例如，心血管疾病和病症、炎性疾病和病症、自身免疫疾病和病症、肺疾病和病症、眼疾病和病症、代谢疾病和病症、神经系统疾病和病症(例如，神经变性疾病和病症)；由衰老引起的年龄相关的疾病和病症；年龄相关的疾病；皮肤疾病和病症；和移植相关的疾病和病症。老化的突出特征是在分子、细胞、组织和生物水平上发生的功能逐渐丧失或退化。年龄相关的退化引起公认的病理，如肌肉萎缩、动脉粥样硬化和心力衰竭、骨质疏松症、肺动脉瓣关闭不全、肾功能衰竭、神经变性(包括黄斑变性、阿尔茨海默病和帕金森病)和许多其他疾病。
在本发明优选的实施方案中，所述的衰老相关疾病或病症为肺疾病和病症，包括但不限于肺纤维化、慢性阻塞性肺疾病、哮喘、囊性纤维化、肺气肿、支气管扩张、和年龄相关肺功能丧失的肺疾病，优选为肺纤维化。
在本发明优选的另一实施方案中，所述的衰老相关疾病或病症为肾脏疾病和病症，所述的肾脏疾病和病症包括肾功能障碍、肾纤维化、肾衰竭。
药学上可接受的载体
本发明用于治疗衰老相关疾病或病症的药物还包括药学上可接受的载体和/或辅料。
术语“药学上可接受的载体”包括适用于制备所需特定剂型的任何和所有溶剂、稀释剂或其他液体媒介物、分散或悬浮助剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、固体粘合剂、润滑剂等。可用作药学上可接受的载体的材料的一些示例包括，但不限于，糖，诸如乳糖、葡萄糖和蔗糖；淀粉，诸如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，诸如羧甲基纤维素钠、乙基纤维素和乙酸纤维素；粉末黄蓍胶；麦芽；明胶；滑石；赋形剂，诸如可可脂和栓剂蜡；油，诸如花生油、棉籽油、红花油、麻油、橄榄油、玉米油和大豆油；二醇，诸如丙二醇；酯，诸如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，诸如氢氧化镁和氢氧化铝；海藻酸；无热原水；等渗盐水；林格氏液；乙醇和磷酸盐缓冲液， 以及根据配制人员的判断，组合物中还可以存在其他无毒相容的润滑剂诸如十二烷基硫酸钠和硬脂酸镁，以及着色剂、释放剂、包衣剂、甜味剂、调味剂和芳香剂、防腐剂和抗氧化剂。
本发明中，可以采用本领域熟知的多种方法来将所述的预防或治疗衰老相关疾病或病症的药物给药于哺乳动物。包括但不限于：皮下注射、肌肉注射、经皮给予、局部给予、植入、缓释给予等。
本发明所述的DBC1的促进剂或ML216(CAS号：1430213-30-1)的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的DBC1的促进剂或ML216的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
细胞
术语“细胞”包括原核细胞和真核细胞。常用的原核细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核细胞包括酵母细胞、昆虫细胞和哺乳动物细胞。较佳地，该细胞是哺乳动物细胞。
作为一种实施方案，所述的哺乳动物细胞为肺细胞，优选为肺成纤维细胞，在本发明的具体实施方案中，所述的肺成纤维细胞为人胚肺成纤维细胞，所述的人胚肺成纤维细胞包括但不仅限于WI-38、KMB17、IMR-90、MRC-5、2BS，优选为IMR-90。
作为一种实施方案，所述的哺乳动物细胞为肾脏细胞，所述的肾脏细胞包括但不限于猴肾CVI细胞系(例如COS-7、ATCC CRL1651)；人胚胎肾系(例如293、293T、293FT)；幼地鼠肾细胞(例如BHK、ATCC CCL10)；猴肾细胞(例如CVI ATCC CCL70)；非洲绿猴肾细胞(例如VERO-76、ATCC CRL-1587)；犬肾细胞(例如MOCK、ATCC CCL34)等。
药物筛选
本发明提供了一种筛选预防或治疗衰老相关疾病或病症的候选药物的方法，所述的方法包括以下步骤：
(1)用待测试物质处理表达或含有DBC1的体系；
(2)检测所述体系中DBC1的表达水平；
(3)选择可上调DBC1表达水平的物质为候选药物。
所述的表达或含有DBC1的体系可以是细胞(或细胞培养物)体系，所述的细胞可 以是内源性表达DBC1的细胞；或可以是重组表达DBC1的细胞。所述的表达或含有DBC1的体系还可以是亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。
除非另有定义，本发明上下文中的所有科技术语具有本领域普通技术人员所理解的相同含义。此外，对部分术语解释如下：
如本文中在诸如“A和/或B”的短语中使用的术语“和/或”旨在包括A和B两者；A或B；A(单独)；以及B(单独)。同样地，在诸如“A、B和/或C”的短语中使用的术语“和/或”旨在涵盖以下实施方案的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；以及C(单独)。
本文中使用的术语“治疗”通常涉及治疗人类或动物(例如，被兽医所应用)，其中可达到某些预期的治疗效果，例如，抑制病症的发展(包括降低发展速度、使发展停止)、改善病症和治愈病症。还包括作为预防措施(例如预防)的治疗。对还没有发展为病症但有发展为该病症危险的患者的用途，也包括在术语“治疗”中。
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1 DBC1缺失对细胞的影响
一、实验方法：
1、为检测DBC1基因敲低或过表达对DNA损伤后293T细胞存活率的影响，使用活细胞计数试剂盒(Cell Counting Kit-8，CCK-8，jd226)对顺铂(50μM)处理后的DBC1基因敲低293T细胞(pRS-DBC1，Origene，TR303704)或DBC1过表达293T细胞(pMSCV-Flag-DBC1，自建质粒)进行细胞存活率分析。为检测DBC1基因敲低或过表达对293T细胞凋亡的影响，使用膜联蛋白V染色法(FITC Annexin V Apoptosis Detection Kit I，BD Biosciences，556547)和流式细胞术定量分析经星形孢菌素(1μM，2小时)处理的DBC1基因敲低细胞或DBC1过表达293T细胞的凋亡水平。采用Western Blot检测DBC1基因在DBC1基因敲低细胞或DBC1过表达293T细胞中的表达水平。
2、采用Western Blot分析经依托泊苷(50μM，24小时)处理的DBC1基因敲低293T细胞中BCL-2、p53和p21蛋白水平。
3、对经依托泊苷(10μM，24小时)处理的DBC1基因敲低293T细胞进行细胞周 期分析。将细胞接种到6孔细胞培养板中，并用10μM依托泊苷处理24小时。然后收获细胞并用PBS洗涤两次。细胞在-20℃的70％预冷乙醇中固定至少一晚。第二天，固定的细胞用PBS洗涤两次并在补充有RNase A(100μg/ml)的50μg/ml PI中在黑暗中染色30分钟。最终细胞由流式细胞术定量分析。
4、使用DNA损伤试剂依托泊苷诱导细胞衰老，该过程为DNA损伤所致衰老(DNA damage induced senescence，DIS)。对经依托泊苷诱导细胞衰老的DBC1基因敲低IMR-90细胞进行SA-β-Gal染色分析，研究DBC1基因敲低对IMR-90细胞衰老的影响，其中，经DMSO处理的细胞为对照组。DIS诱导方法为：早期传代的IMR-90细胞在第16代用依托泊苷(20μM)处理24小时。24小时后培养基替换为新鲜培养基替换细胞，并继续生长7天，在7天内频繁更换新鲜培养基，使IMR-90细胞发生衰老。SA-β-Gal染色为：在室温下将细胞固定在G/F固定剂(含有50％戊二醛和37％甲醛的PBS溶液)中5分钟。在PBS中洗涤两次后，将细胞在染色溶液[含有200mM铁氰化钾、200mM亚铁氰化钾、200mM MgCl 2、6M NaCl和1mg/ml X-gal的柠檬酸盐/钠磷酸盐缓冲液(含有0.1M柠檬酸，0.2M磷酸钠的水溶液)]在37℃无CO 2加湿室中放置2-4小时。最后在显微镜下观察并拍照。
5、RT-PCR分析检测DBC1基因敲低IMR-90细胞中IL-6、IL-8、IL-1α、CXCL-1、CXCL-2、MMP-2、MMP-3和MMP-9的表达水平。
6、在依托泊苷诱导的DNA损伤后的第4天和第7天，进行Ki-67染色分析DBC1基因敲低IMR-90细胞的增殖情况，其中，经DMSO处理的细胞为对照组。
二、实验结果
1、如图1的A所示，敲低DBC1基因可以提高DNA损伤后的细胞存活率；
2、如图1的B所示，DBC1基因敲低细胞中发生凋亡的细胞数较少；
3、如图8的A、图8的B所示，DBC1过表达会提高细胞死亡率以应对DNA损伤，特别是提高细胞凋亡水平。
4、如图1的C所示，DNA损伤后，DBC1基因敲低细胞的凋亡水平降低主要归因于凋亡抑制剂BCL-2的高水平表达，而关键的促凋亡因子p53没有显著改变。
5、p21是一种CDK抑制剂，阻止细胞周期通过G1/S期进展，如图1的D所示，随着p21诱导水平的提高，DBC1基因敲低细胞在DNA损伤后表现出延长的G1期阻滞。
6、如图1的E所示，与对照组比，在DBC1敲低的IMR-90细胞中，不论是否存在DNA损伤，与衰老相关的半乳糖苷酶(SA-β-gal)染色阳性细胞的数量均增加了2-3倍，且衰老相关分泌表型(SASP)因子(如IL-6、IL-8、MMP-3)的表达水平也显著增加(如 图1的F所示)。
6、如图1的G所示，Ki-67染色结果显示，在经依托泊苷诱导DNA损伤后的第4天、第7天，DBC1敲低细胞的增殖水平下降。
上述结果证明，抑制DBC1的表达水平能够促进DNA损伤，进而促进细胞衰老，并减少细胞凋亡。
实施例2 DBC1调控DNA损伤诱导的细胞衰老的机制研究
一、实验方法
1、Western Blot分析经依托泊苷(50μM，24小时)处理的DBC1基因敲低293T细胞中BLM、p16和p21蛋白水平。Western Blot分析经依托泊苷(50μM，24小时)处理的BLM基因敲低293T细胞中DBC1、BLM、p16和p21蛋白水平。
2、对BLM敲低293T细胞的细胞周期进行分析。
3、经依托泊苷诱导DIS后，对BLM敲低IMR-90细胞进行SA-β-Gal染色分析。
4、经依托泊苷(50μM，24小时)诱导DNA损伤后，采用Western Blot分析过表达DBC1的BLM敲低293T细胞中BLM和p21蛋白水平。
5、经依托泊苷诱导DIS后，对过表达DBC1的BLM基因敲低IMR90细胞进行SA-β-Gal染色分析。
6、为了进一步研究DBC1和BLM之间的相关性及其在衰老中的功能，分别对DBC1敲低细胞或BLM敲低293T细胞进行RNA测序分析。如图3的A所示，将DBC1敲低细胞和BLM敲低293T细胞中常见的差异表达基因以维恩图的形式呈现，并对这些差异表达基因进行基因本体论分析，-log10(P值)作为分类的函数，满足P值<0.05。运用KEGG富集分析，与对照细胞相比，DBC1基因敲低细胞和BLM基因敲低细胞的共同差异表达基因。
7、分析依托泊苷处理(50μM，24小时)对DBC1基因敲除293T细胞和BLM基因敲除293T细胞中的衰老相关基因表达水平的影响。
二、实验结果
1、如图2的A所示，在诱导DNA损伤后，与对照组细胞相比，DBC1基因敲低细胞中的BLM蛋白水平下降，其趋势与p21的增加大致成反比。
2、如图2的B所示，当诱导DNA损伤时，与对照组相比，BLM基因敲低细胞的p21表达水平升高，细胞周期G1期延长(如图2的C所示)，p53或p16表达水平没有变化，该结果与DBC1基因敲除细胞相似(如图1的C、图1的D所示)。
3、如图2的D所示，在通过DNA损伤诱导细胞衰老的模式中，与对照细胞相比， BLM缺失的IMR-90细胞的衰老水平升高。
4、如图2的E所示，将DBC1转染到BLM缺失的细胞中，诱导DNA损伤后，随着越多的DBC1存在，越多的BLM蛋白被保留下来，p21的水平也随着降低。
5、在DNA损伤诱导细胞衰老的模式中，将DBC1转染到BLM缺陷的IMR-90细胞中可以减少SA-β-gal阳性细胞的数量，抑制衰老水平(如图2的F所示)。与BCL-2表达水平升高且细胞凋亡水平降低的DBC1敲低细胞(如图1的B和图1的C)不同，敲低BLM对BCL-2的表达水平、细胞凋亡水平均没有影响。
6、分别对DBC1敲低细胞和BLM敲低细胞进行RNA测序，对两组数据中常见变化的转录本进行分析得到具有200个基因重叠的基因表达谱(GEPs)。经生物过程(Biological Process)中的GO富集分析表明，“细胞老化”是两个数据集共有的最富集的上调通路之一(如图3的B所示)。对DBC1和BLM共同调控基因的KEGG分析显示，最富集的通路与氧化磷酸化、亨廷顿病、代谢通路、帕金森病和阿尔茨海默病有关，所有这些都是与衰老相关的疾病，如果功能有缺陷就会导致衰老(如图3的C所示)。在此背景下，比较敲低BLM或DBC1对标志性衰老相关基因的影响的转录分析，结果显示它们在DNA损伤下具有非常相似的作用模式，缺少DBC1或BLM均可促进衰老(如图3的D所示)，这为BLM和DBC1在调节细胞衰老中存在功能重叠提供了证据。
综上，上述结果证明DBC1通过维持BLM丰度调控DNA损伤诱导的细胞衰老。
实施例3 DBC1通过与BLM结合来保护BLM不被降解
一、实验方法
1.为进一步检测DBC1基因敲低293T细胞中BLM蛋白水平下降是否与半胱天冬酶相关，本发明使用泛半胱天冬酶抑制剂Z-VAD(OMe)-FMK对细胞进行处理。对经依托泊苷(100μM)或Z-VAD(OMe)-FMK处理24小时的DBC1基因敲低293T细胞中的BLM和p21蛋白水平进行Western Blot分析。
2.采用免疫共沉淀对DBC1和BLM相互作用进行分析。
3.对DBC1进行一系列截短，将全长或截断DBC1与BLM进行免疫共沉淀实验。全长和截短突变体DBC1(pcDNA3.1-DBC1-V5，pcDNA3.1-DBC1-V5△NT，pcDNA3.1-DBC1-V5△Nudix，自建质粒)的示意结构如图4的C底部图所示。FL代表全长；ΔNudix表示Nudix结构域的缺失(残基339-463)；ΔNT表示N-末端结构域(残基1-263)的缺失。
4.为检测DBC1与BLM的结合是否有助于DBC1保护BLM免受切割，将全长(FL)或N端截短突变体DBC1(ΔNT)的导入到DBC1敲低293T细胞，在经依托泊苷诱导 (50μM，24小时)的DNA损伤后，通过Western Blot分析细胞中的BLM和p21蛋白水平。
二、实验结果
1、如图4的A所示，50μM浓度下，Z-VAD(OME)-FMK可以将DNA损伤后BLM的丢失减少到与对照组相当的水平，p21的水平也相应降低。该结果证明，DBC1的存在减少了DNA损伤激活的半胱天冬酶对BLM的切割。
2、如图4的B所示，DBC1与BLM存在相互作用。进一步确定DBA1中与BLM结合的结构域，实验结果显示，缺少N-端243个氨基酸残基的DBC1突变体不能与LM相互作用，而缺少DBC1中的Nudix结构域对DBC1与BLM的相互作用没有影响(如图4的C所示)。
3、如图4的D所示，与DBC1基因敲低细胞相比，重新引入N端截短的突变体DBC1对BLM的减少没有显著影响，而重新引入全长DBC1会抑制由DNA损伤导致的BLM蛋白的减少，与对照细胞的情况相似。当重新引入全长DBC1时，DNA损伤引起的p21诱导减少，而引入非相互作用突变体没有得到类似的结果。
综上，上述结果证明DBC1通过与BLM结合来保护BLM免受DNA损伤引起的切割。
实施例4 ML216通过促进DBC1-BLM相互作用保护BLM免于降解并抑制DNA损伤诱导的衰老。
一、实验方法
1、用依托泊苷(100μM)、ML216(50μM)或两者同时处理293T细胞24小时后进行免疫共沉淀实验，观察ML216对DBC1-BLM相互作用的影响。
2、用依托泊苷(100μM)或ML216(50μM)处理DBC1基因敲低293T细胞24小时，然后通过Western Blot分析BLM和p21蛋白水平。
3、在不同浓度的ML216下，进行DBC1与BLM的体外免疫沉淀实验。将FLAG-DBC1高表达293T细胞的4等份裂解物分别与0、20、50、100μM ML216孵育12h后，用FLAG抗体进行免疫沉淀实验，采用Western Blot检测BLM蛋白水平。
4、Western Blot检测经不同浓度ML216处理(24小时)的293T细胞中BLM、γH2AX和p21的蛋白水平，以依托泊苷处理组作为阳性对照(100μM，24小时)。
5、用依托泊苷或依托泊苷与不同浓度的ML216共处理诱导IMR90细胞DIS后检测SA-β-Gal阳性细胞数，采用RT-PCR检测诱导后IMR90细胞中p16、p21、IL-1α、IL-6、CXCL-1的表达水平。
6、采用不同浓度ML216处理293T细胞24小时，检测细胞同源重组(HR)修复效率。HR步骤如下：将pcDNA3.1-HR质粒(自建质粒)、pCBASceI质粒(Addgene，26477)和dsRed2-N1质粒(Addgene，54493)转染到细胞中。转染后48小时进行药物处理，最终细胞由流式细胞术定量分析。GFP阳性细胞与RFP阳性细胞的比率用于计算HR修复效率。
7、检测经ML216(50μM，24小时)处理的DBC1基因敲低293T细胞的HR修复效率。
二、实验结果
1、如图5的A所示，经ML216处理后，无论是否存在DNA损伤，更多的BLM蛋白被Flag-DBC1拉下来，而且ML216也增加了input组中BLM蛋白水平。
2、如图5的B所示，在对照细胞中，依托泊苷诱导DNA损伤后，ML216增加了BLM蛋白水平，几乎完全抑制了p21蛋白水平，而在DBC1基因敲除细胞中，BLM蛋白水平增加较少，p21蛋白水平仅被抑制了约50％，该结果显示了ML216对DBC1的功能依赖性。
3、如图5的C所示，结果表明BLM和DBC1与ML216的相互作用呈剂量依赖性增加。
4、如图5的D所示，经ML216处理的细胞中，包括p21、γH2AX在内的衰老标志物的表达水平没有显著变化。但是，在经依托泊苷诱导的DNA损伤中，ML216可以降低p21和γ-H2AX的水平。
5、如图5的E、图5的F所示，与下调衰老标志物p21和γ-H2AX蛋白水平的结果一致，ML216显著降低经依托泊苷诱导DIS的细胞中SA-β-Gal阳性衰老细胞的比例。如图5G所示，ML216还降低了SASP因子IL-1a、IL-6和CXCL1的表达水平。
6、如图5的H所示，ML216处理增加了HR修复效率，而当DBC1被敲低时，ML216处理会不增加HR修复效率(如图5的I所示)，该结果证明ML216对DBC1具有功能依赖性。
综上，上述数据证明，ML216通过促进DBC1-BLM相互作用保护BLM免受降解，从而抑制DNA损伤诱导的细胞衰老。
实施例5 ML216减少特发性肺纤维化(IPF)小鼠模型中与衰老相关的病理变化
一、实验方法
1、单次气管内滴注2mg/kg博莱霉素诱导C57BL/6J小鼠肺部DNA损伤和衰老(如图6A所示)。
2、博莱霉素灌胃一周后，对两组小鼠分别腹腔注射ML216(0.5mg/kg)或赋形剂，每周2次，共3周。3周后检测小鼠的肺功能参数，包括：动态顺应性、峰值顺应性和弦顺应性。进行Masson染色(Masson trichrome staining Kit，Solarbio，G1343)和H&E染色(H&E染色由北京协和医学院医学分子生物学国家重点实验室的组织学系进行)检测肺纤维化状态。采用羟脯氨酸测定小鼠肺组织胶原含量(Hydroxyproline Assay Kit，Solarbio，BC0255)。检测小鼠肺组织纤维化标志物基因的mRNA表达水平。使用ELISA检测小鼠肺和血清中关键的SASP因子(Mouse CXCL1/KC(IL-8)ELISA Kit，Absin，ABS520017；Mouse CCL2/JE/MCP-1ELISAKit，Absin，ABS520016；Mouse IL-6ELISAKit，Absin，ABS520004)。采用SA-β-Gal染色分析小鼠肺部细胞衰老水平。对于组织SA-β-Gal染色，将冷冻切片在冰冷的固定液(含有2％甲醛和0.2％戊二醛的预冷PBS)中固定7分钟。然后用预冷的PBS洗涤样品3次，每次10分钟。冰冻切片用PAP笔包围，染色液染色(同细胞染色液)，在37℃无CO 2加湿室中过夜。最后在显微镜下观察并拍照。
二、实验结果
1、如图6的B所示，博莱霉素的处理使小鼠肺弹性降低20-50％，该结果是通过测量顺应性指数得到的，如动态顺应性、峰值顺应性和弦顺应性等，所有这些指数都经ML216治疗后得到显著改善。
2、如图6的C，Masson染色、H&E染色结果显示，ML216可以减轻IPF小鼠肺纤维化。
3、如图6的D所示，羟脯氨酸测定实验结果显示，ML216组小鼠肺组织胶原含量明显低于对照组。
4、如图6的E所示，ML216组小鼠的肺纤维化标志基因的mRNA表达水平降低。
5、肺组织纤维化显著提高了肺和血清中IL-6、CXCL1和MCP-1的蛋白水平。ML216给药显著降低了肺中IL-6和血清中MCP-1的蛋白水平，而其他因素则呈下降趋势(如图7的A和图7的B所示)。ML216治疗组肺中p21的诱导率显著低于对照组(如图7的C所示)。如图7的D、图7的E所示，ML216处理组小鼠肺部的SA-β-Gal阳性细胞减少，该结果证明ML216处理组小鼠整体衰老状态较对照组小鼠轻。
综上，上述结果证明，ML216给药可以抑制肺细胞衰老来减轻IPF小鼠肺纤维化，进而改善肺功能。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (13)

  1. 一种用于预防和/或治疗衰老相关疾病或病症的试剂，其特征在于，所述试剂为促进DBC1与BLM相互作用的试剂。
  2. 如权利要求1所述的试剂，其中，所述试剂为ML216。
  3. 如权利要求1或2所述的试剂，其中，所述衰老相关疾病或病症选自肺部疾病和病症，以及肾脏疾病和病症；
    优选的，所述肺部疾病和病症选自肺纤维化、慢性阻塞性肺疾病、哮喘、囊性纤维化、肺气肿、支气管扩张和年龄相关肺功能丧失的肺疾病；所述肾脏疾病和病症选自肾功能障碍、肾纤维化和肾衰竭；
    更优选的，所述的肺部疾病和病症为肺纤维化。
  4. 一种药物组合物，所述药物组合物包括DBC1促进剂和如权利要求1～3任一项所述的试剂。
  5. 如权利要求4所述的药物组合物，其特征在于，所述DBC1促进剂选自含有DBC1的表达载体和DBC1蛋白。
  6. 如权利要求5所述的药物组合物，其特征在于，所述表达载体为核酸或细胞。
  7. 如权利要求6所述的药物组合物，其特征在于，所述细胞为哺乳动物细胞；
    优选的，所述哺乳动物细胞选自肺细胞和肾脏细胞；
    更优选的，所述的肺细胞为肺成纤维细胞；所述肾脏细胞为人胚肾细胞；
    进一步更优选的，所述的肺成纤维细胞为人胚肺成纤维细胞，例如为人胚肺成纤维细胞IMR-90；所述人胚肾细胞选自293细胞、293T细胞和293FT细胞，例如为293T细胞。
  8. 如权利要求4～7任一项所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体和/或辅料。
  9. 根据权利要求4～8任一项所述的药物组合物，其特征在于，所述药物组合物的剂型为片剂、胶囊剂、颗粒剂、丸剂、滴丸剂、糖浆剂、粉剂、散剂、栓剂、滴剂、乳剂、注射液、溶液或混悬液。
  10. 一种用于预防和/或治疗衰老相关疾病或病症的药物组合物，其特征在于，所述药物组合物如权利要求4～9任一项所述。
  11. 如权利要求10所述的药物组合物，其特征在于，所述衰老相关疾病或病症如权利要求3所定义。
  12. 一种预防和/或治疗衰老相关疾病或病症的方法，其特征在于，所述方法包括向有需要的患者施用有效量的如权利要求1～3任一项所述的试剂或者如权利要求4～9任一项所述的药物组合物的步骤。
  13. 如权利要求12所述的方法，其特征在于，所述衰老相关疾病或病症如权利要求3所定义。
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