WO2020024774A1 - Dgcr8在制备治疗和/或预防动物骨关节炎产品中的应用 - Google Patents

Dgcr8在制备治疗和/或预防动物骨关节炎产品中的应用 Download PDF

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WO2020024774A1
WO2020024774A1 PCT/CN2019/095174 CN2019095174W WO2020024774A1 WO 2020024774 A1 WO2020024774 A1 WO 2020024774A1 CN 2019095174 W CN2019095174 W CN 2019095174W WO 2020024774 A1 WO2020024774 A1 WO 2020024774A1
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osteoarthritis
dgcr8
stem cells
mesenchymal stem
sequence
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PCT/CN2019/095174
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French (fr)
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曲静
刘光慧
邓丽萍
任若通
刘尊鹏
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中国科学院动物研究所
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the present invention relates to the application of DGCR8 in the field of biomedicine for preparing products for treating and / or preventing animal osteoarthritis.
  • Osteoarthritis (osteoarthritis, OA) is a common degenerative disease with pathological features characterized by degeneration of articular cartilage, increased inflammation and osteophyte formation. Its clinical manifestations are joint pain, swelling, dysfunction, and severe cases lead to joint disability, which seriously affects the quality of life of patients.
  • the occurrence of osteoarthritis is related to many factors, including heredity, age, gender, obesity, trauma, manual labor, endocrine, metabolism, immunity, and so on. Among them, age is the most important cause of osteoarthritis.
  • osteoarthritis has become an important disease that threatens human health.
  • Surgical treatment methods include joint dissection and joint replacement.
  • the former underwent arthroscopic dissection of the ruptured meniscus, which has a certain short-term effect after surgery, but the long-term effect is not ideal.
  • the latter is to remove the cartilage and bone spurs on the articular surface damaged by wear and tear, and then replace metal and ultra-high molecular polyethylene or artificial cartilage as the active surface of the joint at the corresponding site, thereby alleviating pain.
  • the procedure has some short-term and long-term complications, such as infections, looseness and wear of components, and osteolysis.
  • Cell therapy The main pathology of osteoarthritis is the degradation of articular cartilage. Transplanting biologically active chondrocytes into the joint can effectively delay joint pain.
  • MSCs Mesenchymal stem cells
  • Gene therapy As a new type of therapy, gene therapy has many advantages, such as simple operation, long effective period.
  • osteoarthritis the main factor that causes osteoarthritis.
  • various cells in the joint such as chondrocytes, synovial cells, and mesenchymal stem cells all undergo cell aging and functional degradation. Therefore, genes that can delay or resist cellular aging can provide a new idea for the treatment of osteoarthritis. But at present, there are no reports of aging or longevity-related genes as targets for the treatment of osteoarthritis.
  • the object of the present invention is to provide the use of DGCR8 in the preparation of products for treating and / or preventing osteoarthritis in animals.
  • the present invention first provides any one of the following applications of DGCR8: X1, preparing a product for treating and / or preventing animal osteoarthritis; X2, treating and / or preventing animal osteoarthritis; X3, preparing before treating and / or preventing animal Products for osteoarthritis caused by cruciate ligament cut or osteoarthritis caused by physiological aging; X4, treatment and / or prevention of osteoarthritis caused by osteoarthritis or aging of physiologic aging induced by anterior cruciate ligament cut; X5, preparation Products that delay the senescence of mesenchymal stem cells; X6, products that delay the senescence of mesenchymal stem cells; X7, products that delay the replicative senescence of mesenchymal stem cells; X8, products that delay the replicative senescence of mesenchymal stem cells; X9, preparation Product that delays the pathological senescence of mesenchymal stem cells; X10, delays the pathological senescence
  • the DGCR8 may be the following A1) or A2) or A3): A1) the amino acid sequence is the protein of sequence 2; A2) the amino acid sequence of sequence 2 is substituted and / or deleted and / or one or Several amino acid residues have the same function as the protein derived from A1); A3) a fusion protein obtained by attaching a tag to the N-terminus or / and C-terminus of A1) or A2).
  • amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing may be attached with a tag as shown in the following table.
  • DGCR8 in the above A2 is a protein having 75% or more identity with the amino acid sequence of the protein shown in Sequence 2 and having the same function.
  • the identity having 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity .
  • the DGCR8 in the above A2) can be artificially synthesized, or its coding gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding DGCR8 in the above A2) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown at positions 430-2751 of Sequence 1, and / or performing one or several base pairs.
  • a missense mutation and / or a coding sequence obtained by attaching the tag shown in the table above to the 5 'end and / or the 3' end.
  • the DNA molecule shown in positions 430-2751 of sequence 1 encodes DGCR8 shown in sequence 2.
  • the protein described in A2) may be a protein represented by positions 1-773 of sequence 5 in the sequence listing.
  • the present invention also provides any of the following applications of the biological material related to the DGCR8: X1, preparing a product for treating and / or preventing animal osteoarthritis; X2, treating and / or preventing animal osteoarthritis; X3, preparing Products for the treatment and / or prevention of osteoarthritis induced by anterior cruciate ligament cut in an animal or physiological aging; X4, the treatment and / or prevention of osteoarthritis or physiological aging induced by an cruciate ligament cut in an animal Osteoarthritis; X5. Preparation of products to delay the senescence of mesenchymal stem cells; X6. Preparation of products to delay the senescence of mesenchymal stem cells; X7.
  • the biological material is any one of the following B1) to B10): B1 ) A nucleic acid molecule encoding the DGCR8; B2) an expression cassette containing B1) the nucleic acid molecule; B3) a recombinant vector containing the nucleic acid molecule of B1); B4) a recombinant vector containing the expression cassette of B2); B5) Contains B1) The recombinant microorganism of the nucleic acid molecule; B6) the recombinant microorganism containing B2) the expression cassette; B7) the recombinant microorganism containing B3) the recombinant vector; B8) the recombinant microorganism containing B4) the recombinant vector; B9) containing B1) a transgenic cell line of the nucleic acid molecule; B10) a transgenic cell line containing the expression cassette of B2).
  • nucleic acid molecule in B1) may be any one of the following b1) -b4):
  • the coding sequence is a cDNA molecule or DNA molecule at positions 430-2751 of sequence 1 in the sequence listing; b2) a cDNA molecule or DNA molecule at positions 430-2751 of sequence 1 in the sequence listing; b3) and b1) or b2 )
  • a defined nucleotide sequence having 75% or more identity and encoding the DGCR8 cDNA molecule or DNA molecule; b4) under strict conditions with the nucleotide sequence defined by b1) or b2) or b3) Hybridize and encode a cDNA molecule or a DNA molecule of the DGCR8.
  • nucleic acid molecule in b3) is a cDNA molecule or a DNA molecule shown at positions 4658-6979 of sequence 4 in the sequence listing.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding the DGCR8 of the present invention can easily adopt known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the DGCR8 of the present invention.
  • the nucleotide sequence of the invention is equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a natural nucleic acid sequence. “Identity” includes a nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention having 75% or higher, or 85% or higher, or 90% or higher, or 95% or Higher identity nucleotide sequences. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
  • the stringent conditions may be as follows: 50 ° C, hybridization in a mixed solution of 7% sodium lauryl sulfate (SDS), 0.5M NaPO 4 and 1mM EDTA, at 50 ° C, 2 ⁇ SSC, 0.1 Rinse in% SDS; also: 50 ° C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, rinse in 50 ° C, 1 ⁇ SSC, 0.1% SDS; also: 50 ° C , Hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse at 50 ° C, 0.5 ⁇ SSC, 0.1% SDS; also: 50 ° C, 7% SDS, 0.5M NaPO 4 and Hybridize in a mixed solution of 1mM EDTA, rinse at 50 ° C, 0.1 ⁇ SSC, 0.1% SDS; also: hybridize at 50 ° C in a mixed solution of 7%
  • the aforementioned 75% or more identity may be 80%, 85%, 90%, or 95% identity.
  • the expression cassette containing the nucleic acid molecule encoding the DGCR8 refers to DNA capable of expressing the DGCR8 in a host cell, and the DNA may include
  • the promoter for transcription of the DGCR8 gene may further include a terminator for terminating the transcription of the DGCR8 gene.
  • the expression cassette may further include an enhancer sequence.
  • Recombinant vectors containing the DGCR8 gene expression cassette can be constructed using existing expression vectors.
  • the vector may be a plasmid, a cosmid, a phage, or a viral vector.
  • the plasmid may specifically be a pLE4 vector.
  • the recombinant vector may specifically be pLE4-DGCR8 or pLE4-DGCR8-mtDRBD.
  • the pLE4-DGCR8 is a recombinant vector obtained by ligating a DNA molecule shown in positions 430-2751 of Sequence 1 to a pLE4 vector digested with BamH1 and Mlu1 by means of homologous recombination.
  • the sequence of the pLE4-DGCR8 may be sequence 3 in the sequence listing, and the pLE4-DGCR8 can express a full-length DGCR8 protein comprising amino acids 1-773 in sequence 2.
  • the pLE4-DGCR8-mtDRBD is obtained by mutating the codons of Ala at position 568-569 of the sequence encoding DGCR8 in pLE4-DGCR8 to the codons of Lys, Lys, and changing the codons of Ala at 676-677.
  • the codon was mutated into a recombinant vector obtained from the codons of Lys and Lys.
  • the sequence of pLE4-DGCR8-mtDRBD may be sequence 4 in the sequence listing.
  • the pLE4-DGCR8-mtDRBD can express the protein shown in Sequence 5.
  • the microorganism may be a virus, yeast, bacteria, algae or fungus.
  • the virus may be a lentivirus.
  • the recombinant microorganism may be a recombinant lentivirus.
  • the recombinant lentivirus may specifically be a lentivirus packaged with the pLE4-DGCR8 or the pLE4-DGCR8-mtDRBD.
  • the transgenic cell line may or may not include propagation material.
  • the animal may be c1) or c2): c1) mammal; c2) human or mouse.
  • the DGCR8 or the biological material also belongs to the protection scope of the present invention.
  • the present invention also provides a product having any of the following functions of Y1-Y5, the product containing the DGCR8 or the biological material: Y1, treating and / or preventing animal osteoarthritis; Y2, treating and / or preventing animal Anterior cruciate ligament cut-induced osteoarthritis or osteoarthritis caused by physiological aging; Y3, delay the senescence of mesenchymal stem cells; Y4, delay the replicative senescence of mesenchymal stem cells; Y5, delay the pathology of mesenchymal stem cells Sexual aging.
  • the present invention also provides a method for treating and / or preventing osteoarthritis in an animal, the method comprising: administering the DGCR8 to an animal having osteoarthritis to achieve the treatment and / or prevention of osteoarthritis.
  • the osteoarthritis may be osteoarthritis induced by anterior cruciate ligament amputation or osteoarthritis caused by physiological aging.
  • the pathological senescence of the mesenchymal stem cells may be senescence caused by mutation of the adult premature senescence gene.
  • the adult progeria gene may be a WRN gene.
  • the treatment and / or prevention of animal osteoarthritis may be specifically embodied in any of the following aspects: C1) reducing the degradation of articular cartilage; C2) promoting the proliferation of joint cells (such as chondrocytes); C3) delaying the aging of joint cells; C4 ) Reduce the inflammatory response in the joint cavity; C5) reduce the apoptosis of joint cells.
  • the mesenchymal stem cells may be mesenchymal stem cells that are all positive for CD73, CD90, and CD105.
  • the senescence of the mesenchymal stem cells may be manifested as a growth retardation of the mesenchymal stem cells.
  • the senescence of the mesenchymal stem cells may be specifically manifested as an increase in SA- ⁇ -gal activity in the mesenchymal stem cells.
  • the product may be a drug or a vaccine.
  • Figure 1 DGCR8 and its C-terminal mutant can delay the replicative senescence of human mesenchymal stem cells.
  • A Expression of DGCR8 in young (2nd generation) and senescent (12th generation) human mesenchymal stem cells;
  • B After infection with Luc, DGCR8, and DGCR8-mtDRBD lentivirus in 6th generation human mesenchymal stem cells, Detection of SA- ⁇ -gal activity continued to the 12th passage;
  • C After infected with Luc, DGCR8, and DGCR8-mtDRBD lentivirus in human mesenchymal stem cells of the 6th passage, the cell proliferation molecular markers continued to the 12th passage Ki67 expression detection;
  • D 6th generation human mesenchymal stem cells were infected with Luc, DGCR8 and DGCR8-mtDRBD lentivirus respectively, and then continued to culture until the 12th generation of cell proliferation detection;
  • E 6th generation human mesenchymal
  • FIG. 2 DGCR8 and its C-terminal mutant can delay the accelerated aging phenotype of human mesenchymal stem cells carrying mutations in the human adult progeria gene.
  • A DGCR8 expression in WT and WS mesenchymal stem cells of the 7th generation;
  • B Luc, DGCR8, and DGCR8-mtDRBD lentivirus infection in human mesenchymal stem cells of the 6th generation, respectively, and continue to culture to the 12th generation of SA - ⁇ -gal activity detection;
  • C After the sixth generation of human mesenchymal stem cells were infected with Luc, DGCR8, and DGCR8-mtDRBD lentivirus, respectively, continue to culture until the twelfth generation of cell proliferation ability detection;
  • D in the sixth generation of human Mesenchymal stem cells were infected with Luc, DGCR8, and DGCR8-mtDRBD lentiviruses, respectively, and cultured to the 12th passage of the cell
  • Figures 3 and 4 Therapeutic effects of DGCR8 and its C-terminal mutant gene in mouse osteoarthritis induced by anterior cruciate ligament resection.
  • A schematic diagram of the experimental process
  • B micro-CT results and bone density test results of mouse joints after gene therapy using Luc, DGCR8 and DGCR8-mtDRBD lentivirus
  • C Luc, DGCR8 and DGCR8-mtDRBD
  • lentivirus gene therapy cartilage staining results of mouse joint sections, and pathological grading results of mouse arthritis according to the standards of the International Society for Osteoarthritis Research (Society International).
  • the cell culture conditions in the following examples are all 37 ° C and 5% CO 2 .
  • the mouse used in the present invention is a product of Beijing Huafukang Biotechnology Co., Ltd., and is bred in an animal welfare facility of the Institute of Biophysics, which has a room temperature of 23 ° C, a 12-hour day and night cycle, and can freely eat and drink. All animal experiments were approved by the Animal Welfare Committee of the Institute of Biophysics, Chinese Academy of Sciences.
  • the cell lines used in the present invention are as follows:
  • the human embryonic stem cell H9 cell line is a product of WiCell, article number: WA09 (H9) -DL-7.
  • the human embryonic kidney cell 293T line is an ATCC product with the product number CRL-3216.
  • the mesenchymal stem cells carrying human adult progeria gene mutations are mesenchymal stem cells with loss of WRN function in the Chinese patent (Pluripotent Stem Cells Carrying Human Adult Progeria Gene Mutations and Preparation Method, ZL201510137846.4), An inventor is deposited at the Institute of Biophysics, Chinese Academy of Sciences, and is publicly available from the applicant.
  • the cell culture medium used in the present invention is as follows:
  • Human embryonic stem cell H9 cell culture medium CDF12 76.9% (volume percentage, v / v) DMEM / F12 (ThermoFisher, 11330-032), 20% (volume percentage, v / v) Knock-out serum substitute ( Gibco, 10828-028), 0.1% (volume percent, v / v) b-mercaptoethanol (Invitrogen, 21985-023), 1% (volume percent, v / v) GlutaMAX (Invitrogen, 35050-061), 1% (Volume percentage, v / v) non-essential amino acids (Invitrogen, 11140-050), 1% (volume percentage, v / v) penicillin / streptomycin (Invitrogen, 15070-063), 10ng / mL recombinant human fibroblasts Growth factor (JPC, bFGF).
  • JPC bFGF
  • Mouse embryo fibroblast medium 87% (volume percentage, v / v) DMEM (Hyclone), 10% (volume percentage, v / v) fetal bovine serum (Gibco, 10100- 147), 1% (volume percent, v / v) non-essential amino acids (Invitrogen, 11140-050), 1% (volume percent, v / v) GlutaMAX (Invitrogen, 35050-061), 1% (volume percent, v / v) Penicillin / streptomycin (Invitrogen, 15070-063).
  • Embryonic kidney cell 293T line medium 87% (volume percentage, v / v) DMEM (Hyclone), 10% (volume percentage, v / v) fetal bovine serum (Gibco, 10100-147), 1% ( Volume percent, v / v) Non-essential amino acids (Invitrogen, 11140-050), 1% (volume percent, v / v) GlutaMAX (Invitrogen, 35050-061), 1% (volume percent, v / v) penicillin / chain Mycin (Invitrogen, 15070-063).
  • Mesenchymal stem cell differentiation medium 88% (volume percentage, v / v) ⁇ -MEM + GlutaMAX (Invitrogen, 12571071), 10% (volume percentage, v / v) fetal bovine serum (Gibco, 10100- 147), 1% (volume percentage, v / v) non-essential amino acids (Invitrogen, 11140-050), 1% (volume percentage, v / v) penicillin / streptomycin (Invitrogen, 15070-063), 10ng / mL FGF2 (Joint, Protein, Central), 5ng / ml, TGF ⁇ (Humanzyme, HZ1131).
  • Mesenchymal stem cell culture medium 89% (volume percentage, v / v) ⁇ -MEM + GlutaMAX (Invitrogen, 12571071), 10% (volume percentage, v / v) fetal bovine serum (Gibco, 10100-147 ), 1% (volume percentage, v / v) penicillin / streptomycin (Invitrogen, 15070-063), 1ng / ml recombinant human fibroblast growth factor (JPC, FGF2).
  • the biological materials used in the lentivirus packaging of the present invention are as follows:
  • Lentiviral packaging vectors are psPAX2 (Addgene, # 12260) and pMD2G (Addgene, # 12259).
  • the lentiviral vector is pLE4 (a gift from Dr. Tomoaki and Hishida) (Ren et al., Visualization of aging-associated chromatin alternatives with an engineered TALE system, Cell Research (2017) 27: 483-504.) With the consent of Tomoaki and Hishida The biological material is obtained from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the cell culture method according to the present invention is as follows:
  • Digestion method Digestion with collagenase IV (Gibco) for 3-5 minutes, then digestion enzymes are aspirated, washed with DMEM / F12 basal medium (ThermoFisher, 11330-032) twice, and then added with CDF12 medium, and transferred with glass The liquid tube scraped the clones into small pieces and inoculated them onto the paved MEF feeder layer.
  • Fluorescein FITC-labeled anti-human cell surface recognition molecule CD90 antibody (555595), BD Biosciences.
  • Fluorescein PE-labeled anti-human cell surface recognition molecule CD73 antibody (550257), BD Biosciences.
  • Fluorescein APC-labeled isotype control antibody (555751), BD Biosciences.
  • Fluorescein PE-labeled isotype control antibody (555749), BD Biosciences.
  • Fluorescein FITC-labeled isotype control antibody (555742), BD Biosciences.
  • Anti-human DGCR8 antibody (ab191875), Abcam.
  • SA- ⁇ -gal senescence-associated beta-galactosidase
  • SA- ⁇ -gal is a hydrolytic enzyme in lysosomes, which has enhanced activity in senescent cells.
  • the enzyme can hydrolyze it and make cells Appears blue.
  • SA- ⁇ -gal has thus become a "gold standard" for rapid and rapid detection of cellular senescence.
  • the specific method is as follows:
  • Step 2) After completion, add 2mL of staining solution (40mM citric acid / sodium phosphate buffer, 5mM K 4 [Fe (CN) 6 ], 5mM K 3 [Fe (CN) 6 ], 150mM NaCl, 2mM MgCl 2. 1 mg / mL X-gal), and incubate overnight in a 37 ° C bacterial incubator protected from light.
  • staining solution 40mM citric acid / sodium phosphate buffer, 5mM K 4 [Fe (CN) 6 ], 5mM K 3 [Fe (CN) 6 ], 150mM NaCl, 2mM MgCl 2. 1 mg / mL X-gal
  • step 3 wash with PBS twice, observe under an inverted microscope, and take a picture.
  • the experimental data in the present invention are expressed as mean ⁇ standard deviation and analyzed by GraghPad Prism 5 statistical software, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001.
  • Example 1 Delaying effect of DGCR8 and its C-terminal mutant on senescence of mesenchymal stem cells
  • human mesenchymal stem cells differentiated from human embryonic stem cells can be continuously passaged from the first generation to the twelfth generation under in vitro culture conditions.
  • the mesenchymal stem cells of the 12th generation showed obvious growth retardation, the cell volume became larger, and the morphology of the cells was flat.
  • the second and twelfth generation of mesenchymal stem cells were used as test cells, where the second generation cells represent young cells and the twelfth generation cells represent senescent cells.
  • the total protein of the two types of cells was extracted, and the test cells were lysed with RIPA cell lysate (Biyuntian, P0013B), and then the protein concentration in the cells was measured using the BCA protein quantification kit (Biyuntian, P0010). Finally, 20 ⁇ g of protein was taken for immunization Blot experiment.
  • the primary antibody was DGCR8 antibody (Abcam, ab191875)
  • the secondary antibody was goat anti-rabbit IgG / HRP (Zhongshan Golden Bridge, ZDR-5306)
  • the internal reference was ⁇ -tubulin (Santa Cruz, 5274)
  • the secondary antibody was goat anti-mouse IgG / HRP (Nakasugi Golden Bridge, ZDR-5307).
  • the results are shown in A in FIG. 1, and the expression of DGCR8 protein was significantly down-regulated in the 12th generation (senescent) mesenchymal stem cells compared with the 2nd generation (young) mesenchymal stem cells.
  • Lentiviruses encoding Luc, DGCR8, or a C-terminal mutant thereof were infected with mesenchymal stem cells of the sixth generation as test cells.
  • the specific method is as follows:
  • DGCR8 GenBank: NM_022720.6, updated 09-MAY-2018
  • NCBI NCBI
  • positions 430-2751 of Sequence 1 are the coding sequence (CDS) of the DGCR8 protein
  • sequence of the DGCR8 protein is Sequence 2 in the Sequence Listing.
  • primers were designed to perform PCR amplification of the coding sequence of DGCR8 using the total cDNA of human mesenchymal stem cells as a template.
  • the primer sequences are as follows:
  • DGCR8-CDS-F 5′-atggagacagatgagagcccctctccgctc-3 ′ (sequence 6 in the sequence listing)
  • DGCR8-CDS-R 5′-tcacacgtccacggtgcacaggggctcac-3 ′ (sequence sequence 7)
  • the coding sequence of DGCR8 (positions 430-2751 of Sequence 1) obtained by PCR was ligated to pLE4 vector digested with BamH1 and Mlu1 by homologous recombination, and the resulting sequence was correctly named as a lentiviral plasmid encoding DGCR8. It is pLE4-DGCR8.
  • the sequence of pLE4-DGCR8 is sequence 3 in the sequence listing.
  • PLE4-DGCR8 can express the full-length DGCR8 protein containing amino acids 1-773 in sequence 2.
  • sequence of -mtDRBD, pLE4-DGCR8-mtDRBD is sequence 4 in the sequence listing
  • pLE4-DGCR8-mtDRBD can express the protein shown in sequence 5
  • the difference between this protein and the protein expressed by pLE4-DGCR8 is: pLE4-DGCR8-mtDRBD expression
  • the protein is a protein obtained by mutating Ala at positions 568-569 of DGCR8 sequence expressed by pLE4-DGCR8 to Lys, and mutating Ala at positions 676-677 to Lys.
  • Solution A 600 ⁇ L Opti-MEM (Gibco) and 18 ⁇ L Lipo3000
  • Solution B 600 ⁇ L Opti-MEM, 15 ⁇ g pLE4-DGCR8 or pLE4-DGCR8-mtDRBD, 10 ⁇ g psPAX2, and 5 ⁇ g pMD2.G, respectively, and then add 30 ⁇ L P3000), after mixing thoroughly, add the B liquid dropwise to the A liquid, shake while adding, and then mix thoroughly. After incubating at room temperature for 10-15 minutes, the mixed solution was added dropwise to 293T cells (10 cm petri dishes), gently shaken, and cultured at 37 ° C and 5% CO 2 .
  • lentiviruses encoding DGCR8 and DGCR8 C-terminal mutants were obtained, which are respectively referred to as DGCR8 lentivirus and DGCR8-mtDRBD lentivirus.
  • the sixth-generation mesenchymal stem cells were used as test cells, and the lentivirus encoding the DGCR8 or the C-terminal mutant thereof prepared as described above was used for infection.
  • the specific method is: 0.2 ⁇ L of lentivirus encoding DGCR8 or a C-terminal mutant thereof and 2 ⁇ L of Polybrene are added to a culture well (one well of a 6-well plate) inoculated with 6th generation mesenchymal stem cells. The medium was changed the next day, followed by normal culture and passage. At the same time, the same conditions were used to infect the sixth-generation mesenchymal stem cells with lentivirus expressing firefly luciferase (Luc) as a control treatment.
  • the virus is Flag-luciferase in the article "SIRT6 safeguards human mesenchymal cells from oxidative stress by coactivating NRF2.Pan et.al ,. Cell research. (2016) 26: 190-205.”
  • lentivirus encoding Luc, DGCR8 or a C-terminal mutant thereof After being infected with a lentivirus encoding Luc, DGCR8 or a C-terminal mutant thereof, the mesenchymal stem cells were continuously passaged to passages 10-12.
  • Cell senescence markers such as SA- ⁇ -gal staining, cell proliferation molecular markers (Ki67) detection, and DNA damage response molecular markers ( ⁇ -H2AX and 53BP1) and other aging biological research methods have shown that the results are slower than those encoding Luc.
  • lentiviruses encoding DGCR8 or its C-terminal mutant were able to significantly delay the replicative senescence of mesenchymal stem cells (BF in Figure 1).
  • the expression of DGCR8 protein is detected in mesenchymal stem cells (WS mesenchymal stem cells, human mesenchymal stem cells carrying human adult senescence gene mutations) carrying mutations in the human adult senility syndrome. Variety. Compared with normal mesenchymal stem cells, WS mesenchymal stem cells have a phenotype of accelerated senescence due to the deletion of the disease-causing gene WRN. Immunoblot results (A in Figure 2) showed that the expression of DGCR8 in WS mesenchymal stem cells was significantly lower than that of normal mesenchymal stem cells.
  • 1.4 DGCR8 and its C-terminal mutant can delay the accelerated aging phenotype of mesenchymal stem cells carrying mutations in the human premature aging gene
  • the fourth-generation WS mesenchymal stem cells were used as test cells to infect the lentiviruses encoding Luc, DGCR8, or their C-terminal mutants, respectively.
  • the method for preparing and infecting the lentivirus encoding Luc, DGCR8 or its C-terminal mutant is the same as that in step 1.2.
  • this example uses the DGCR8 encoding lentivirus obtained in Example 1 or the C-terminal mutant encoding DGCR8 Lentivirus for Gene Therapy in Anterior Cruciate Ligament Cut-induced Osteoarthritis Mice
  • mice used were all SPF, male, 10 weeks old, weighing 20 ⁇ 2g, Beijing Huafukang Biotechnology Co., Ltd.
  • Experimental equipment and materials include: isoflurane gas anesthesia apparatus, epilator, surgical scissors, scalpel, Venus scissors, sutures, penicillin, stereoscope, 20 ⁇ L micro-injector.
  • mice were divided into a non-surgical group (6, non-surgical) and a surgical group (18).
  • the specific experimental steps for establishing a mouse model of osteoarthritis by anterior cruciate ligament resection in the surgery group are as follows:
  • mice are anesthetized, and the legs of the mice are depilated using a depilator.
  • Penicillin was applied to the surgical site to prevent infection, and a mouse model of osteoarthritis induced by anterior cruciate ligament cut was obtained.
  • Lentivirus-mediated gene therapy was performed 7 days after surgery to establish a mouse model of osteoarthritis induced by anterior cruciate ligament cut ( Figure 3A).
  • the lentivirus encoding DGCR8 or the DGCR8 C-terminal mutant obtained in Example 1 was resuspended in 20 ⁇ L of mesenchymal stem cell culture medium for treatment, and the expression of firefly luciferase (luciferase, Luc) in Example 1 Lentivirus was used as a treatment control.
  • the left and right knee joint cavities of the osteoarthritis mice in the surgery group were injected with 10 ⁇ L of lentivirus encoding Luc, DGCR8 lentivirus or DGCR8-mtDRBD lentivirus, respectively, and 6 of each type of lentivirus were injected.
  • the left and right knee joint cavities of the mice in the non-surgical group were injected with 6 l of lentivirus coding for Luc, respectively.
  • the effect of DGCR8 and its C-terminal mutant gene on osteoarthritis was evaluated. The specific method is as follows:
  • mice injected with Luc lentivirus showed obvious trauma in the joints after surgery.
  • the joints of mice injected with DGCR8 lentivirus or DGCR8-mtDRBD lentivirus in the surgical group were non-surgical.
  • the surgery group was similar and no obvious injury was seen (Figure 3B).
  • Hematoxylin (Nakasugi Golden Bridge, ZLI-9610) was stained for 8 minutes, and rinsed under running water for 2 minutes.
  • mice were sacrificed at the seventh week after virus injection, and the right leg and knee joints of each mouse were removed for RNA extraction and qPCR detection. It was found that compared with the non-surgical group, the control group injected with Luc expressing was slower.
  • the expression of senescence markers P16 and P21 in mouse joints was significantly increased, while the expressions of inflammatory factors IL6 and MMP13 were also significantly increased, suggesting that obvious cellular senescence and inflammatory reactions will occur during the onset of osteoarthritis.
  • the experiments of the present invention prove that DGCR8 and its C-terminal mutant gene have a clear function of delaying the senescence of mesenchymal stem cells, and that DGCR8 and its C-terminal mutant gene have a clear therapeutic effect in osteoarthritis diseases.
  • the invention provides a new idea for the development of gene therapy for osteoarthritis, and expands the selectable range of clinical gene therapy.

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Abstract

本发明公开了DGCR8在制备治疗和/或预防动物骨关节炎产品中的应用。本发明发现，DGCR8具有如下功能：治疗和/或预防动物骨关节炎；治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；延缓间充质干细胞的衰老；延缓间充质干细胞的复制性衰老；延缓间充质干细胞的病理性衰老。实验证明，DGCR8具有明确的延缓间充质干细胞衰老的功能，并且在骨关节炎疾病中具有明确的治疗效果，为开发用于骨关节炎的基因治疗提供了新的思路，且扩大了临床基因治疗的可选择范围。

Description

DGCR8在制备治疗和/或预防动物骨关节炎产品中的应用 技术领域
本发明涉及生物医学领域中，DGCR8在制备治疗和/或预防动物骨关节炎产品中的应用。
背景技术
骨关节炎(osteoarthritis，OA)是一种常见的退行性病变，病理特征为关节软骨的退化，炎症的增加和骨赘的形成。其临床表现为关节疼痛，肿胀，功能障碍，严重者导致关节残疾，严重影响了患者的生活质量。骨关节炎的发生与多种因素有关，包括遗传，年龄，性别，肥胖，外伤，体力劳动，内分泌，代谢，免疫等等。其中，年龄是骨关节炎最主要的发病因素。随年龄的增长，关节软骨发生退行性变化，软骨基质中的粘多糖含量减少，基质丧失硫酸软骨素，纤维成分增加，软骨的韧性减低。随着人口老龄化问题日益严峻，骨关节炎已是威胁人类健康的重要疾病。
目前，骨关节炎的治疗有多种方法，包括药物，手术及新兴的细胞治疗(cell therapy)和基因治疗(gene therapy)。1.药物治疗：临床上的常用药物有透明质酸制剂和糖皮质激素。透明质酸钠是关节腔滑液的主要成分，在关节中起到润滑作用。通过关节腔注入后可明显改善滑液组织的炎症反应，增强关节液的粘稠性和润滑功能，保护关节软骨，促进关节软骨的愈合与再生，缓解疼痛，增加关节的活动度。2.手术治疗：对于症状严重，药物治疗无效的，则需采用手术治疗。手术治疗方法包括关节清扫和关节置换。前者在关节镜下对破裂的半月板进行清扫，术后近期有一定的疗效，但远期效果则并不理想。后者则是将磨损破坏的关节面软骨、骨刺等去除，然后在相应的部位换上金属和超高分子聚乙烯或者人工软骨作为关节的活动面，从而缓解疼痛。该手术存在一定的近期和远期并发症，如感染，部件的松动和磨损、骨溶解。3.细胞治疗：基于骨关节炎的主要病理为关节软骨的退化，向关节内移植有生物活性的软骨细胞可有效延缓关节病痛。间充质干细胞(Mesenchymal stem cells，MSC)具有向软骨分化的潜能，同时具有抗炎，抗免疫特性，因此也广泛用于骨关节炎的细胞治疗。4.基因治疗：作为一种新型的疗法，基因治疗具有很多优势，例如简单易操作，有效时期长。
已有研究表明引发骨关节炎的最主要的因素是衰老。随着衰老，关节内的多种细胞如软骨细胞，滑膜细胞，间充质干细胞均发生细胞衰老和功能退化。因此，能够延缓或抵抗细胞衰老的基因可以为骨关节炎的治疗提供一种新的思路。但目前，还没有以衰老或长寿相关基因作为靶点来治疗骨关节炎的报道。
发明公开
本发明的目的是提供DGCR8在制备治疗和/或预防动物骨关节炎产品中的应用。
本发明首先提供了DGCR8的下述任一应用：X1、制备治疗和/或预防动物骨关节炎的产品；X2、治疗和/或预防动物骨关节炎；X3、制备治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎的产品；X4、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；X5、制备延缓间充质干细胞的衰老的产品；X6、延缓间充质干细胞的衰老；X7、制备延缓间充质干细胞的复制性衰老的产品；X8、延缓间充质干细胞的复制性衰老；X9、制备延缓间充质干细胞的病理性衰老的产品；X10、延缓间充质干细胞的病理性衰老。
上述应用中，所述DGCR8可为如下A1)或A2)或A3)：A1)氨基酸序列为序列2的蛋白质；A2)在序列2的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
为了使A1)中的蛋白质便于纯化，可在由序列表中序列2所示的氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如下表所示的标签。
表：标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述A2)中的DGCR8，为与序列2所示蛋白质的氨基酸序列具有75％或75％以上同一性且具有相同功能的蛋白质。所述具有75％或75％以上同一性为具有75％、具有80％、具有85％、具有90％、具有95％、具有96％、具有97％、具有98％或具有99％的同一性。
上述A2)中的DGCR8可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述A2)中的DGCR8的编码基因可通过将序列1的第430-2751位所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上上表所示的标签的编码序列得到。其中，序列1的第430-2751位所示的DNA分子编码序列2所示的DGCR8。
上述应用中，A2)所述蛋白质可为序列表中序列5的第1-773位所示的蛋白质。
本发明还提供了与所述DGCR8相关的生物材料的下述任一应用：X1、制备治疗和/或预防动物骨关节炎的产品；X2、治疗和/或预防动物骨关节炎；X3、制备治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的 骨关节炎的产品；X4、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；X5、制备延缓间充质干细胞的衰老的产品；X6、延缓间充质干细胞的衰老；X7、制备延缓间充质干细胞的复制性衰老的产品；X8、延缓间充质干细胞的复制性衰老；X9、制备延缓间充质干细胞的病理性衰老的产品；X10、延缓间充质干细胞的病理性衰老；所述生物材料为下述B1)至B10)中的任一种：B1)编码所述DGCR8的核酸分子；B2)含有B1)所述核酸分子的表达盒；B3)含有B1)所述核酸分子的重组载体；B4)含有B2)所述表达盒的重组载体；B5)含有B1)所述核酸分子的重组微生物；B6)含有B2)所述表达盒的重组微生物；B7)含有B3)所述重组载体的重组微生物；B8)含有B4)所述重组载体的重组微生物；B9)含有B1)所述核酸分子的转基因细胞系；B10)含有B2)所述表达盒的转基因细胞系。
上述应用中，B1)所述核酸分子可为如下b1)-b4)中的任一种：
b1)编码序列是序列表中序列1的第430-2751位的cDNA分子或DNA分子；b2)序列表中序列1的第430-2751位的cDNA分子或DNA分子；b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码所述DGCR8的cDNA分子或DNA分子；b4)在严格条件下与b1)或b2)或b3)限定的核苷酸序列杂交，且编码所述DGCR8的cDNA分子或DNA分子。
上述应用中，b3)所述核酸分子为序列表中序列4的第4658-6979位所示的cDNA分子或DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码所述DGCR8的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的所述DGCR8的核苷酸序列75％或者更高同一性的核苷酸，只要编码所述DGCR8且具有所述DGCR8功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列2所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述应用中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC， 0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述应用中，B2)所述的含有编码所述DGCR8的核酸分子的表达盒(所述DGCR8基因表达盒)，是指能够在宿主细胞中表达所述DGCR8的DNA，该DNA不但可包括启动所述DGCR8基因转录的启动子，还可包括终止所述DGCR8基因转录的终止子。进一步，所述表达盒还可包括增强子序列。
可用现有的表达载体构建含有所述DGCR8基因表达盒的重组载体。
上述应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒具体可为pLE4载体。
B2)所述重组载体具体可为pLE4-DGCR8或pLE4-DGCR8-mtDRBD。所述pLE4-DGCR8为将序列1的第430-2751位所示的DNA分子通过同源重组的方式连接到经过BamH1和Mlu1酶切的pLE4载体上得到的重组载体。所述pLE4-DGCR8的序列可为序列表中序列3，所述pLE4-DGCR8能表达包含序列2中第1-773位氨基酸的DGCR8全长蛋白。
所述pLE4-DGCR8-mtDRBD为将所述pLE4-DGCR8中编码DGCR8序列的第568-569位的Ala Ala的密码子分别突变为Lys Lys的密码子、并将第676-677位的Ala Ser的密码子突变为Lys Lys的密码子得到的重组载体。所述pLE4-DGCR8-mtDRBD的序列可为序列表中序列4。所述pLE4-DGCR8-mtDRBD能表达序列5所示的蛋白质。
上述应用中，所述微生物可为病毒、酵母、细菌、藻或真菌。所述病毒可为慢病毒。
B5)所述重组微生物可为重组慢病毒。所述重组慢病毒具体可为利用所述pLE4-DGCR8或所述pLE4-DGCR8-mtDRBD包装得到的慢病毒。
上述应用中，所述转基因细胞系可包括也可不包括繁殖材料。
上述应用中，所述动物可为c1)或c2)：c1)哺乳动物；c2)人或小鼠。
所述DGCR8或所述生物材料，也属于本发明的保护范围。
本发明还提供了具有如下Y1-Y5中任一功能的产品，所述产品含有所述DGCR8或所述生物材料：Y1、治疗和/或预防动物骨关节炎；Y2、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；Y3、延缓间充质干细胞的衰老；Y4、延缓间充质干细胞的复制性衰老；Y5、延缓间充质干细胞的病理性衰老。
本发明还提供了治疗和/或预防动物骨关节炎的方法，所述方法包括：对患有骨关节炎的的动物施用所述DGCR8，实现骨关节炎的治疗和/或预防。
所述骨关节炎可为前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎。
本发明中，所述间充质干细胞的病理性衰老可为成年早衰症基因突变引发的衰老。所述成年早衰症基因可为WRN基因。
所述治疗和/或预防动物骨关节炎具体可体现在如下任一方面：C1)降低关节软骨的退化；C2)促进关节细胞(如软骨细胞)的增殖；C3)延缓关节细胞的衰老；C4)降低关节腔的炎症反应；C5)降低关节细胞的凋亡。
所述间充质干细胞可为CD73、CD90和CD105均为阳性的间充质干细胞。
所述间充质干细胞的衰老可表现为间充质干细胞的生长阻滞。所述述间充质干细胞的衰老具体可表现为间充质干细胞中SA-β-gal活性的增加。
所述产品可为药物或疫苗。
附图说明
图1：DGCR8及其C端突变体能够延缓人间充质干细胞的复制性衰老。A：年轻(第2代)和衰老(第12代)的人间充质干细胞中DGCR8的表达情况；B：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的SA-β-gal活性检测；C：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的细胞增殖分子标志物Ki67表达检测；D：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的细胞增殖能力检测；E：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的DNA损伤反应检测。
图2：DGCR8及其C端突变体能够延缓携带人类成年早衰症基因突变的人间充质干细胞的加速衰老表型。A：第7代WT和WS间充质干细胞中DGCR8的表达情况；B：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的SA-β-gal活性检测；C：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的细胞增殖能力检测；D：在第6代人间充质干细胞中分别感染Luc、DGCR8和DGCR8-mtDRBD慢病毒后，继续培养至第12代的细胞衰老分子标志物RT-qPCR检测。
图3和图4：DGCR8及其C端突变体基因在前交叉韧带切断手术诱导的小鼠骨关节炎中的治疗效果。图3中，A：实验流程示意图；B：利用Luc、DGCR8和DGCR8-mtDRBD慢病毒进行基因治疗后，小鼠关节micro-CT结果和骨密度检测结果；C：利用Luc、DGCR8和DGCR8-mtDRBD慢病毒进行基因治疗后，小鼠关节切片后的软骨染色结果，以及根据国际骨关节炎研究学会(The Osteoarthritis Research Society International)的标准进行的小鼠关节炎病理评级结果。图 4中，D：利用Luc、DGCR8和DGCR8-mtDRBD慢病毒进行基因治疗后，小鼠关节RNA测序的差异基因表达热图分析结果；E：利用Luc、DGCR8和DGCR8-mtDRBD慢病毒进行基因治疗后，对小鼠关节RNA进行炎症、凋亡和软骨、成骨发生相关基因的RT-qPCR检测结果。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸，末位均为相应DNA的3′末端核苷酸。下述实施例中的细胞培养条件如无特殊说明，均为37摄氏度，5％CO 2。本发明中使用的小鼠为北京华阜康生物科技股份有限公司产品，饲养于室温23℃，12小时昼夜周期并且可以自由摄食和饮水的生物物理研究所动物福利设施中。所有的动物实验均获得中国科学院生物物理研究所动物福利委员会的批准。
1.本发明用到的细胞系如下：
人胚胎干细胞H9细胞系是WiCell公司产品，货号：WA09(H9)-DL-7。
人胚胎肾细胞293T系为ATCC产品，货号CRL-3216。
携带人类成年早衰症基因突变的间充质干细胞，为中国专利(携带人类成年早衰症基因突变的多能干细胞及制备方法，ZL201510137846.4)中的WRN功能丧失的间充质干细胞，并由第一发明人保存在中国科学院生物物理研究所，公众可从申请人处获得。
2.本发明用到的细胞培养基如下：
(1)人胚胎干细胞H9细胞培养基CDF12：76.9％(体积百分比，v/v)DMEM/F12(ThermoFisher,11330-032)，20％(体积百分比，v/v)Knock-out血清替代物(Gibco,10828-028)，0.1％(体积百分比，v/v)b-巯基乙醇(Invitrogen，21985-023)，1％(体积百分比，v/v)GlutaMAX(Invitrogen，35050-061)，1％(体积百分比，v/v)非必需氨基酸(Invitrogen，11140-050)，1％(体积百分比，v/v)青霉素/链霉素(Invitrogen，15070-063)，10ng/mL重组人成纤维细胞生长因子(JPC，bFGF)。
(2)小鼠胚胎成纤维细胞培养基(MEF培养基)：87％(体积百分比，v/v)DMEM(Hyclone)，10％(体积百分比，v/v)胎牛血清(Gibco,10100-147)，1％(体积百分比，v/v)非必需氨基酸(Invitrogen，11140-050)，1％(体积百分比，v/v)GlutaMAX(Invitrogen，35050-061),1％(体积百分比，v/v)青霉素/链霉素(Invitrogen，15070-063)。
(3)胚胎肾细胞293T系培养基：87％(体积百分比，v/v)DMEM(Hyclone)，10％(体积百分比，v/v)胎牛血清(Gibco,10100-147)，1％(体积百分比， v/v)非必需氨基酸(Invitrogen，11140-050)，1％(体积百分比，v/v)GlutaMAX(Invitrogen，35050-061),1％(体积百分比，v/v)青霉素/链霉素(Invitrogen，15070-063)。
(4)间充质干细胞分化培养基：88％(体积百分比，v/v)α-MEM+GlutaMAX(Invitrogen，12571071)，10％(体积百分比，v/v)胎牛血清(Gibco,10100-147)，1％(体积百分比，v/v)非必需氨基酸(Invitrogen，11140-050)，1％(体积百分比，v/v)青霉素/链霉素(Invitrogen，15070-063)，10ng/mL FGF2(Joint Protein Central)，5ng/ml TGFβ(Humanzyme，HZ1131)。
(5)间充质干细胞培养基：89％(体积百分比，v/v)α-MEM+GlutaMAX(Invitrogen，12571071),10％(体积百分比，v/v)胎牛血清(Gibco,10100-147),1％(体积百分比，v/v)青霉素/链霉素(Invitrogen，15070-063),1ng/ml重组人成纤维细胞生长因子(JPC，FGF2)。
3.本发明中慢病毒包装所用生物材料如下：
慢病毒包装载体是psPAX2(Addgene,#12260)和pMD2G(Addgene,#12259)。
慢病毒载体为pLE4(由Dr Tomoaki Hishida馈赠)(Ren et al.,Visualization of aging-associated chromatin alterations with an engineered TALE system,Cell Research(2017)27:483-504.)经Tomoaki Hishida同意后公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
4.本发明涉及的细胞培养方法如下：
(1)人胚胎干细胞H9的培养方法：人胚胎干细胞系H9(WiCell Research)培养在经丝裂霉素处理的小鼠胚胎成纤维细胞(MEF)上，以CDF12作为培养基，每天更换新鲜培养基，一般5-7天传代一次。传代前一天，用MEF培养基在明胶(Sigma)包被的板子上铺MEF饲养层细胞，待细胞贴壁后，吸去MEF培养基，换成CDF12培养基。传代的方法包括机械法和消化法。机械法：使用毛细玻璃管对人胚胎干细胞克隆进行分块儿，挑取状态良好的克隆转移至铺好的MEF饲养层上。消化法：用消化酶胶原酶IV(Gibco)消化3-5分钟后吸走消化酶，用DMEM/F12基础培养基(ThermoFisher,11330-032)洗两次，再加入CDF12培养基，用玻璃移液管将克隆刮成小块接种到铺好的MEF饲养层上。
(2)间充质干细胞的分化和培养：将人胚胎干细胞进行拟胚体(EB)分化，分化3天，将EB接种于基质胶(matrigel)(Invitrogen公司)包被的6孔板中用上述的间充质干细胞分化培养基进行培养，继续培养2周至纤维状细胞出现。再经过一次传代后，利用流式细胞术分选其中的CD73、CD90和CD105均为阳性的细胞类群，即为高纯度的人间充质干细胞。纯化的间充质干细胞的培养用上述的间充质干细胞培养基进行培养，4-5天传一次代。每次传代以1×10 5细胞密度接种到6孔板中的一个孔中。
4.用于流式细胞术分选间充质干细胞的荧光标记抗体：
荧光素FITC标记的抗人细胞表面识别分子CD90抗体(555595)，BD Biosciences。
荧光素PE标记的抗人细胞表面识别分子CD73抗体(550257)，BD Biosciences。
荧光素APC标记的抗人细胞表面识别分子CD105抗体(17-1057-42)，BD Biosciences。
荧光素APC标记同型对照抗体(555751)，BD Biosciences。
荧光素PE标记同型对照抗体(555749)，BD Biosciences。
荧光素FITC标记同型对照抗体(555742)，BD Biosciences。
5.用于免疫印迹抗体:
抗人DGCR8抗体(ab191875)，Abcam。
抗人β-tubulin抗体(sc-15407)，Santa Cruz Biotechnology。
抗人GAPDH抗体(sc-25778)，Santa Cruz Biotechnology。
6.本发明中检测细胞衰老的SA-β-gal染色
SA-β-gal(senescence-associated beta-galactosidase)是溶酶体中的一种水解酶，在衰老细胞中活性增强，当添加底物X-Gal时，该酶可将其酶解并使细胞显现蓝色。SA-β-gal也因此成为一种快捷迅速检测细胞衰老的“金标准”。具体方法如下：
1)以1×10 5/孔的密度将间充质干细胞接种到明胶(sigma)包被的6孔板的一个孔中，第2天进行染色。
2)步骤1)完成后用固定液(2％(体积百分比，v/v)甲醛+0.2％(体积百分比，v/v)戊二醛+97.8％(体积百分比，v/v)PBS)固定细胞4分钟(一定不要超过5分钟)，用PBS洗2遍。
3)步骤2)完成后每孔加入2mL染色液(40mM柠檬酸/磷酸钠缓冲液、5mM K 4[Fe(CN) 6]、5mM K 3[Fe(CN) 6]、150mM NaCl、2mM MgCl 2、1mg/mL X-gal)，在37℃细菌培养箱避光孵育过夜。
4)步骤3)完成后用PBS洗2遍后，倒置显微镜下观察，拍照。
7.本发明中的实验数据以平均值±标准差表示，用GraghPad Prism 5统计软件分析，*P<0.05,**P<0.01,***P<0.001。
实施例1、DGCR8及其C端突变体对间充质干细胞衰老的延缓作用
1.1伴随间充质干细胞的复制性衰老，DGCR8表达明显下调
利用体外定向分化技术，从人胚胎干细胞分化得到的人间充质干细胞可在体外培养条件下从第1代连续传代到第12代。第12代的间充质干细胞出现明显的生长阻滞，细胞体积变大，形态扁平等细胞衰老表型。以第2代和第12代的间充质干细胞作为供试细胞，其中第2代的细胞代表年轻的细胞，第12代的细胞代表衰老的细胞。提取两种细胞的总蛋白，使用RIPA细胞裂解液(碧云天， P0013B)裂解供试细胞，然后使用BCA蛋白定量试剂盒(碧云天，P0010)测定细胞中蛋白浓度，最后分别取20μg蛋白进行免疫印迹实验。使用一抗为DGCR8抗体(Abcam，ab191875)，二抗为羊抗兔IgG/HRP(中杉金桥，ZDR-5306)，内参为β-tubulin(Santa Cruz,5274)，二抗为羊抗鼠IgG/HRP(中杉金桥，ZDR-5307)。结果如图1中A所示，与第2代(年轻)间充质干细胞相比，DGCR8蛋白的表达在第12代(衰老)间充质干细胞中明显下调。
1.2过表达DGCR8或其C端突变体可延缓间充质干细胞的复制性衰老
以第6代的间充质干细胞作为供试细胞，进行编码Luc、DGCR8或其C端突变体的慢病毒的感染。具体方法如下：
1.2.1制备编码Luc、DGCR8或其C端突变体的慢病毒
根据NCBI提供的DGCR8的cDNA序列(GenBank：NM_022720.6，updated on 09-MAY-2018)，如序列表中序列1所示。其中，序列1的第430-2751位为DGCR8蛋白的编码框(coding sequence，CDS)，DGCR8蛋白的序列为序列表中序列2。基于此序列，设计引物，以人间充质干细胞的总cDNA为模板，进行DGCR8的编码序列的PCR扩增。
引物序列如下：
DGCR8-CDS-F：5′-atggagacagatgagagcccctctccgctc-3′(序列表中序列6)
DGCR8-CDS-R：5′-tcacacgtccacggtgcacaggggctcac-3′(序列表中序列7)
将PCR得到的DGCR8的编码序列(序列1的第430-2751位)通过同源重组的方式连接到经过BamH1和Mlu1酶切的pLE4载体上，将得到的序列正确的编码DGCR8的慢病毒质粒命名为pLE4-DGCR8，pLE4-DGCR8的序列为序列表中序列3，pLE4-DGCR8能表达包含序列2中第1-773位氨基酸的DGCR8全长蛋白。
随后，利用Fast MultiSite Mutagenesis System(北京全式金生物技术有限公司)对构建的慢病毒质粒pLE4-DGCR8同时进行4个氨基酸位点突变，最终获得编码DGCR8 C端突变体的慢病毒质粒pLE4-DGCR8-mtDRBD，pLE4-DGCR8-mtDRBD的序列为序列表中序列4，pLE4-DGCR8-mtDRBD能表达序列5所示的蛋白质，该蛋白与pLE4-DGCR8表达的蛋白质的区别为：pLE4-DGCR8-mtDRBD表达的蛋白质为将pLE4-DGCR8表达的DGCR8序列的第568-569位的Ala Ala突变为Lys Lys、并将第676-677位的Ala Ser突变为Lys Lys得到的蛋白质。
编码Luc、DGCR8或其C端突变体的慢病毒包装：
1)使用Lipo3000转染试剂盒(Thermo Fisher)，将纯化的慢病毒质粒pLE4-DGCR8或pLE4-DGCR8-mtDRBD与包装质粒psPAX2(Addgene,#12260)和pMD2.G(Addgene,#12259)共同转染人胚胎肾细胞293T系。具体如下：分别配制A液(600μL Opti-MEM(Gibco)和18μL Lipo3000)和B液(600μL Opti-MEM、15μg pLE4-DGCR8或pLE4-DGCR8-mtDRBD、10μg psPAX2和5μg pMD2.G， 再加入30μL P3000)，充分混匀后，将B液逐滴加入A液中，边加边振荡，而后充分混匀。室温孵育10-15分钟后，将混合液滴加到293T细胞中(10cm培养皿)，轻轻摇匀，37摄氏度，5％CO 2条件培养。
2)转染8小时后更换新鲜的293T细胞培养基(胚胎肾细胞293T系培养基)继续培养。
3)更换新鲜培养基后的第48-72小时收取培养上清。
4)使用0.45μm滤器过滤培养上清于新的离心管中，4℃离心，转速为19400rpm/分钟，时间2.5小时。
5)离心后，将上清倒掉。使用100μL间充质干细胞培养基重悬沉淀，分装冻存于-80℃。即得到编码DGCR8和编码DGCR8 C端突变体的慢病毒，分别记为DGCR8慢病毒和DGCR8-mtDRBD慢病毒。
1.2.2编码DGCR8或其C端突变体的慢病毒感染间充质干细胞
以第6代的间充质干细胞作为供试细胞，用上述制备好的编码DGCR8或其C端突变体的慢病毒进行感染。具体方法为：0.2μL编码DGCR8或其C端突变体的慢病毒和2μL Polybrene加到接种有第6代的间充质干细胞的培养孔(6孔板的一个孔)中。次日换液，之后正常培养，传代。同时，利用相同条件在第6代的间充质干细胞中感染了表达萤火虫荧光素酶(luciferase，Luc)的慢病毒作为对照处理。该病毒为“SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2.Pan et.al,.Cell research.(2016)26:190-205.”一文中的Flag-luciferase。
感染编码Luc、DGCR8或其C端突变体的慢病毒后，将间充质干细胞连续传代至10-12代。细胞衰老标志物SA-β-gal染色、细胞增殖分子标志物(Ki67)检测以及DNA损伤反应分子标志物(γ-H2AX和53BP1)等衰老生物学研究方法检测的结果表明，与编码Luc的慢病毒相比，编码DGCR8或其C端突变体的慢病毒能够明显延缓间充质干细胞的复制性衰老(图1中B-F)。
1.3 DGCR8或其C端突变体在携带人类成年早衰症基因突变的间充质干细胞的病理性衰老中表达下降
按照步骤1.1的方法，在携带人类成年早衰症(Werner syndrome)基因突变的间充质干细胞(WS间充质干细胞，携带人类成年早衰症基因突变的间充质干细胞)中，检测DGCR8蛋白的表达变化。WS间充质干细胞因携带致病基因WRN的缺失，与正常间充质干细胞相比，表现出加速衰老的表型。免疫印迹结果(图2中A)显示，WS间充质干细胞中的DGCR8的表达量明显低于正常间充质干细胞。
1.4 DGCR8及其C端突变体能够延缓携带人类成年早衰症基因突变的间充质干细胞的加速衰老表型
以第4代的WS间充质干细胞为供试细胞，分别感染编码Luc、DGCR8或其C端突变体的慢病毒。编码Luc、DGCR8或其C端突变体的慢病毒制备方法和感染方法同步骤1.2。
感染病毒后，经过2-3次传代，感染Luc的WS间充质干细胞生长完全阻滞，并且细胞衰老标志物SA-β-gal染色、细胞增殖能力检测以及P16、IL6和LaminB1等衰老分子标志物RT-qPCR检测结果表明，与编码Luc的慢病毒相比，编码DGCR8或其C端突变体的慢病毒能够明显延缓WS间充质干细胞的病理性衰老进程(图2中B-D)。
实施例2.DGCR8及其C端突变体基因对于前交叉韧带切断建立的骨关节炎小鼠的治疗作用
由于实施例1获得的编码DGCR8慢病毒或DGCR8-mtDRBD慢病毒在体外具有良好的延缓间充质干细胞衰老的特性，本实施例将实施例1获得的编码DGCR8慢病毒或编码DGCR8 C端突变体的慢病毒用于前交叉韧带切断诱导的骨关节炎小鼠的基因治疗。
2.1建立前交叉韧带切断诱导的骨关节炎小鼠模型
所用小鼠均为SPF级，雄性，10周，体重20±2g，北京华阜康生物科技股份有限公司。实验器械和材料包括：异氟烷气体麻醉仪，脱毛器，手术剪刀，手术刀，维纳斯剪刀，缝合线，青霉素，体式镜，20μL微量进样器。
小鼠分为非手术组(6只，不进行手术)和手术组(18只)。对手术组小鼠进行前交叉韧带切断术建立骨关节炎小鼠模型的具体实验步骤如下：
1)小鼠麻醉，用脱毛器将小鼠腿部进行脱毛。
2)在小鼠腿部膝盖处剪皮，露出关节部位。
3)将小鼠转移到体式镜下进行手术：经膝关节内侧髌旁切口.逐层分离打开关节腔.将髌骨向外侧脱位后屈膝下完全暴露前交叉韧带(较细的一条白带)，用维纳斯剪在韧带中段予以切断，并剪除2mm韧带组织防止粘连愈合。两条腿的关节部位均进行手术。
4)生理盐水冲洗关节腔.将髌骨复位，严密缝合关节腔及皮肤。
5)在手术部位施加青霉素，防感染，得到前交叉韧带切断诱导的骨关节炎小鼠模型。
2.2 DGCR8及其C端突变体基因对于骨关节炎小鼠的治疗效果评价
在建立前交叉韧带切断诱导的骨关节炎小鼠模型的手术后的第7天，进行慢病毒介导的基因治疗(图3A)。将实施例1获得的编码DGCR8慢病毒或编码DGCR8 C端突变体的慢病毒重悬于20μL间充质干细胞培养基中进行治疗，同时实施例1中表达萤火虫荧光素酶(luciferase，Luc)的慢病毒作为治疗对照。向手术组的骨关节炎小鼠的左右膝关节腔中分别注射10μL编码码Luc的慢病毒、DGCR8慢病毒或DGCR8-mtDRBD慢病毒，每种类型的慢病毒注射6只。向非手术组的小鼠的左右膝关节腔中分别注射10μL编码码Luc的慢病毒注射6只。在注射病毒后的第7周，对DGCR8及其C端突变体基因治疗骨关节炎的效果进行了评价。具体方法如下：
1)micro-CT检测小鼠关节微结构
在注射病毒后的第7周，对各组小鼠，利用micro-CT(PE公司)检测观察关节的微结构。结果显示，与非手术组相比，手术后注射Luc慢病毒的小鼠关节处出现了明显的损伤性空洞；而手术组注射了DGCR8慢病毒或DGCR8-mtDRBD慢病毒的小鼠关节则与非手术组近似，未见明显损伤(图3B)。
2)小鼠关节的软骨组织染色
小鼠骨关节炎的表型之一是关节处软骨细胞的减少。因此，在注射病毒后的第7周处死小鼠，取下每只小鼠左腿膝关节进行软骨的石蜡切片染色。具体步骤如下：
a)取下小鼠左腿膝关节，将肌肉和脂肪剥离干净。
b)用4％多聚甲醛固定72小时。
c)用甲酸脱钙液(5％甲酸，5％甲醛，90％水)进行关节的脱钙处理，时间为7天。
d)脱钙完全后，进行脱水，石蜡包埋，切片。切片厚度为5m。
e)对切片进行脱蜡，复水。
f)铁苏木素(中杉金桥，ZLI-9610)染色8分钟，流水冲洗2分钟。
g)0.02％快绿(Sigma，S2255)染色8分钟，1％醋酸快速漂洗15秒。
h)0.1％番红O(Sigma，F7252)染色5分钟。
i)脱水，封片，镜下观察。
结果显示，与非手术组相比，手术组注射Luc慢病毒的小鼠股骨和胫骨处的软骨细胞明显减少，而当注射DGCR8慢病毒或DGCR8-mtDRBD慢病毒后软骨细胞的比例又有所增加(图3C)。对于染色的切片，根据国际骨关节炎研究学会(The Osteoarthritis Research Society International)的骨关节炎病理评级标准(Osteoarthritis score)进行评级打分，与注射表达Luc的慢病毒相比，注射DGCR8慢病毒或DGCR8-mtDRBD慢病毒小鼠的关节炎程度有明显下降(图3C)。
3)小鼠关节的RNA测序和RT-qPCR分析
小鼠骨关节炎的表型还包括关节腔炎症的增加，细胞的衰老，细胞的凋亡。因此，本发明在注射病毒后第7周处死小鼠，取下每只小鼠右腿膝关节进行了RNA的提取和qPCR检测,发现与非手术组相比，手术组注射表达Luc的对照慢病毒的小鼠关节中衰老标志物P16和P21表达明显增加，同时炎症因子IL6和MMP13表达也明显增加，提示骨关节炎发病过程中会发生明显的细胞衰老和炎症反应，而注射DGCR8慢病毒或DGCR8-mtDRBD慢病毒后则会明显降低上述衰老标志物和炎症因子的表达水平，说明DGCR8和DGCR8-mtDRBD的表达均能够有效缓解骨关节炎的疾病进程(图4D)。随后，本发明对小鼠关节提取的RNA进行了全转录组测序。测序结果显示，与非手术组相比，手术组注射表达Luc慢病毒的小鼠关节细胞的基因表达谱发生明显变化，而手术组注射DGCR8慢病毒或 DGCR8-mtDRBD慢病毒的小鼠关节细胞的基因表达谱则更近似非手术对照组(图4E)。通过RT-qPCR验证，表明手术组注射Luc慢病毒的小鼠关节细胞的炎症和细胞凋亡水平均增加，而软骨和成骨发生相关基因(Runx2,Satb2,Col1a1,Col1a2,Col11a1,Col18a1,Chst3,Chst13,Chsy1)表达则明显下调。手术后注射DGCR8慢病毒或DGCR8-mtDRBD慢病毒则会提高软骨和成骨发生相关基因(Runx2,Satb2,Col1a1,Col1a2,Col11a1,Col18a1,Chst3,Chst13,Chsy1)的表达，同时降低炎症和细胞凋亡(图4E)。该结果证明DGCR8及其C端突变体基因对骨关节炎具有良好的治疗效果。
工业应用
本发明的实验证明DGCR8及其C端突变体基因具有明确的延缓间充质干细胞衰老的功能，并且DGCR8及其C端突变体基因在骨关节炎疾病中具有明确的治疗效果。本发明为开发用于骨关节炎的基因治疗提供了新的思路，且扩大了临床基因治疗的可选择范围。

Claims (10)

  1. DGCR8的下述任一应用：
    X1、制备治疗和/或预防动物骨关节炎的产品；
    X2、治疗和/或预防动物骨关节炎；
    X3、制备治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎的产品；
    X4、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；
    X5、制备延缓间充质干细胞的衰老的产品；
    X6、延缓间充质干细胞的衰老；
    X7、制备延缓间充质干细胞的复制性衰老的产品；
    X8、延缓间充质干细胞的复制性衰老；
    X9、制备延缓间充质干细胞的病理性衰老的产品；
    X10、延缓间充质干细胞的病理性衰老。
  2. 根据权利要求1所述的应用，其特征在于：所述DGCR8为如下A1)或A2)或A3)：
    A1)氨基酸序列为序列2的蛋白质；
    A2)在序列2的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；
    A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
  3. 根据权利要求2所述的应用，其特征在于：A2)所述蛋白质为序列表中序列5的第1-773位所示的蛋白质。
  4. 与权利要求1-3中任一所述DGCR8相关的生物材料的下述任一应用：
    X1、制备治疗和/或预防动物骨关节炎的产品；
    X2、治疗和/或预防动物骨关节炎；
    X3、制备治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎的产品；
    X4、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；
    X5、制备延缓间充质干细胞的衰老的产品；
    X6、延缓间充质干细胞的衰老；
    X7、制备延缓间充质干细胞的复制性衰老的产品；
    X8、延缓间充质干细胞的复制性衰老；
    X9、制备延缓间充质干细胞的病理性衰老的产品；
    X10、延缓间充质干细胞的病理性衰老；
    所述生物材料为下述B1)至B10)中的任一种：
    B1)编码权利要求1-3中任一所述DGCR8的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体；
    B4)含有B2)所述表达盒的重组载体；
    B5)含有B1)所述核酸分子的重组微生物；
    B6)含有B2)所述表达盒的重组微生物；
    B7)含有B3)所述重组载体的重组微生物；
    B8)含有B4)所述重组载体的重组微生物；
    B9)含有B1)所述核酸分子的转基因细胞系；
    B10)含有B2)所述表达盒的转基因细胞系。
  5. 根据权利要求4所述的应用，其特征在于：B1)所述核酸分子为如下b1)-b4)中的任一种：
    b1)编码序列是序列表中序列1的第430-2751位的cDNA分子或DNA分子；
    b2)序列表中序列1的第430-2751位的cDNA分子或DNA分子；
    b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求1-3中任一所述DGCR8的cDNA分子或DNA分子；
    b4)在严格条件下与b1)或b2)或b3)限定的核苷酸序列杂交，且编码权利要求1-3中任一所述DGCR8的cDNA分子或DNA分子。
  6. 根据权利要求5所述的应用，其特征在于：b3)所述核酸分子为序列表中序列4的第4658-6979位所示的cDNA分子或DNA分子。
  7. 根据权利要求1-6中任一所述的应用，其特征在于：所述动物为c1)或c2)：
    c1)哺乳动物；
    c2)人或小鼠。
  8. 权利要求3中所述DGCR8或权利要求4-6中任一所述生物材料。
  9. 具有如下Y1-Y5中任一功能的产品，含有权利要求1-3中任一所述DGCR8或权利要求4-6中任一所述生物材料：
    Y1、治疗和/或预防动物骨关节炎；
    Y2、治疗和/或预防动物前交叉韧带切断诱导的骨关节炎或生理性衰老导致的骨关节炎；
    Y3、延缓间充质干细胞的衰老；
    Y4、延缓间充质干细胞的复制性衰老；
    Y5、延缓间充质干细胞的病理性衰老。
  10. 治疗和/或预防动物骨关节炎的方法，包括：对患有骨关节炎的的动物施用权利要求1-3中任一所述DGCR8，实现骨关节炎的治疗和/或预防。
PCT/CN2019/095174 2018-08-02 2019-07-09 Dgcr8在制备治疗和/或预防动物骨关节炎产品中的应用 WO2020024774A1 (zh)

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CN108904781A (zh) * 2018-08-02 2018-11-30 中国科学院动物研究所 Dgcr8在制备治疗和/或预防动物骨关节炎产品中的应用

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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108904781A (zh) * 2018-08-02 2018-11-30 中国科学院动物研究所 Dgcr8在制备治疗和/或预防动物骨关节炎产品中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AIKO SHIOHAMA: "Molecular Cloning and Expression Analysis of a Novel Gene DGCR8 Located in the DiGeorge Syndrome Chromosomal Region", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 304, no. 1, 31 December 2003 (2003-12-31), pages 184 - 190, XP055684870 *
DATABASE Nucleotide 17 March 2007 (2007-03-17), SHIMIZU, N.: "Homo sapiens DGCR8 gene, complete cds", XP055684874, retrieved from NCBI Database accession no. AB050770.1 *
ZHU, CHANGHONG: "SUM Oylation of DGCR8 and Its Function in miRNA Pathway", CHINESE DOCTORAL DISSERTATION FULL-TEXT DATABASE (BASIS SCIENCES), 15 February 2017 (2017-02-15), XP055684880 *

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