WO2023011489A1 - 一种基因修饰干细胞及其用途 - Google Patents

一种基因修饰干细胞及其用途 Download PDF

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WO2023011489A1
WO2023011489A1 PCT/CN2022/109763 CN2022109763W WO2023011489A1 WO 2023011489 A1 WO2023011489 A1 WO 2023011489A1 CN 2022109763 W CN2022109763 W CN 2022109763W WO 2023011489 A1 WO2023011489 A1 WO 2023011489A1
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cartilage repair
cells
nucleic acid
stem cells
factor
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French (fr)
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薛冰华
于婷婷
张振利
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北京吉源生物科技有限公司
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Definitions

  • the invention relates to genetic engineering, in particular to the technical field of stem cell therapy, in particular to genetically modified stem cells for cartilage repair.
  • Stem cells are a type of cells with self-renewal and differentiation potential, and are an important means for the research or treatment of tumors, cardiovascular diseases and other malignant diseases.
  • Stem cell therapy has great research and application value in the three major fields of life sciences, new drug testing and disease research. It has been widely used in the fields of medical regenerative cell replacement therapy and drug screening, and has become the focus of world attention and research.
  • stem cell drugs are mainly knee osteoarthritis and diabetic foot.
  • Stem cell clinical treatment and research diseases involve all major systems of the body, including: acute myocardial infarction, cerebral palsy in children, premature ovarian failure, psoriasis, interstitial lung disease, knee osteoarthritis, Parkinson's disease, retinitis pigmentosa, age Associated macular degeneration, ulcerative colitis, bone repair, empty nose syndrome, infertility, lupus nephritis, neuromyelitis optica, thin endometrium, pulmonary hypertension due to COPD, decompensated hepatitis B sclerosis, neuropathic pain, meniscal injury, etc.
  • the types of stem cells used in stem cell therapy include: embryonic stem cell-derived cells, neural stem cells, mesenchymal stem cells from various sources (such as fat, umbilical cord, bone marrow, placenta, etc.), and bronchial basal layer cells.
  • the treatment methods involved include: the use of cells alone, the use of cells in combination with materials, and the use of cells in combination with drugs.
  • modifying mesenchymal stem cells with anti-inflammatory factors and factors promoting cartilage differentiation at the same time can not only effectively promote the differentiation of mesenchymal stem cells into cartilage, but also through various The combination of factors comprehensively acts on various types of immune cells and inhibits the activity of various types of immune cells, thus solving the local inflammatory environment of cartilage repair in bone knees and other parts, so that clinical patients can reduce or reduce the risk of using mesenchymal stem cells for bone knee repair. Swelling occurs, thereby completing the present invention.
  • the present invention provides the following aspects:
  • a genetically modified stem cell for cartilage repair therapy includes nucleic acid encoding an anti-inflammatory factor and a nucleic acid encoding a cartilage repair factor.
  • the anti-inflammatory factors are selected from the family of interleukins and TGF ⁇ receptors, preferably, the anti-inflammatory factors are selected from interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Two or more of interleukin-13 (IL-13) and interleukin-37 (IL-37), more preferably interleukin 10 and interleukin 37.
  • the anti-inflammatory factors are selected from the family of interleukins and TGF ⁇ receptors, preferably, the anti-inflammatory factors are selected from interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Two or more of interleukin-13 (IL-13) and interleukin-37 (IL-37), more preferably interleukin 10 and interleukin 37.
  • cartilage repair factor is selected from fibroblast growth factor, FGF2a
  • the cartilage repair factor is selected from fibroblast growth factor 18 (FGF18).
  • the nucleic acid encoding the anti-inflammatory factor is carried by a strong promoter that is easy to be silenced in the stem cell, and the nucleic acid encoding the cartilage repair factor adopts a method that is not easy to be expressed in the stem cell Carried by a medium-silenced medium-strength promoter.
  • genetically modified stem cells for cartilage repair therapy as described above, nucleic acids encoding anti-inflammatory factors and nucleic acid encoding cartilage repair factors linked.
  • a method for preparing gene-modified stem cells for cartilage repair therapy including linking nucleic acids encoding anti-inflammatory factors and nucleic acids encoding cartilage repair factors, and introducing them into mesenchymal stem cells.
  • Step 1 obtaining the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor, digesting the nucleic acid and connecting with the carrier plasmid to obtain the recombinant plasmid;
  • Step 2 transfecting the recombinant plasmid and the packaging plasmid into lentiviral packaging cells to obtain a recombinant lentiviral vector containing nucleic acids encoding anti-inflammatory factors and nucleic acids encoding cartilage repair factors;
  • Step 3 transfecting the recombinant lentiviral vector obtained above into mesenchymal stem cells to obtain genetically modified stem cells for cartilage repair therapy.
  • the ninth aspect there is provided a use of the above-mentioned genetically modified stem cells or the genetically modified stem cells prepared by the above-mentioned method in the preparation of repairing/treating drugs for cartilage diseases.
  • cartilage diseases include degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, hyperosteogeny, rheumatoid arthritis and rheumatoid arthritis Arthritis etc.
  • the genetically modified stem cells provided by the present invention can simultaneously express anti-inflammatory factors and cartilage repair factors, have a good synergistic effect, not only can effectively promote the differentiation of mesenchymal stem cells into cartilage, but also can inhibit the activity of various immune cells, Reduce or avoid local inflammation during cartilage repair;
  • the genetically modified stem cells provided by the present invention not only maintain the function of mesenchymal stem cells, but also can well express lipid anti-inflammatory factors and cartilage repair factors, and can be safely administered to human subjects without triggering immunity primary reaction;
  • the genetically modified stem cells provided by the present invention have the ability to significantly improve the inflammation of cartilage repair in patients' knees, effectively alleviate the problems of reducing/avoiding swelling when using mesenchymal stem cells in bone knee repair, and have great clinical significance. value.
  • Fig. 1 shows the pCDH-103718 plasmid plasmid map of embodiment
  • Fig. 2 shows the phenotype result figure of MSC-103718 cells in the experimental example
  • Figure 3 shows a schematic diagram of the detection results of the expression levels of IL10, IL37 and FGF18 in MSC-103718 cells in the experimental example
  • Figure 4 shows a schematic diagram of the test results of the adipogenic differentiation ability of each sample in the experimental example induced differentiation culture for 21 days;
  • Figure 5 shows a schematic diagram of the detection results of osteogenic differentiation ability of each sample induced differentiation culture for 21 days in the experimental example
  • Figure 6 shows an enlarged schematic diagram of the detection results of the chondrogenic differentiation ability of each sample induced differentiation culture for 21 days in the experimental example
  • Figure 7 shows a schematic diagram of the test results of chondrogenic differentiation ability of each sample induced differentiation culture for 21 days in the experimental example
  • Figure 8 shows a schematic diagram of the regulatory effect of MSC-103718 cells on immune cells
  • Fig. 9 is a schematic diagram showing the results of MSC-103718 cells inhibiting the secretion of TNF- ⁇ from lymphocytes.
  • the inventors jointly modified mesenchymal stem cells with nucleic acids encoding inflammatory factors and nucleic acids encoding cartilage repair factors, which exhibited a significant synergistic effect of multiple genes, which has great clinical value.
  • the genetically modified stem cells are resistant to nucleic acids encoding inflammatory factors and nucleic acids encoding cartilage repair factors.
  • the anti-inflammatory factor is selected from interleukin and TGF ⁇ receptor family, preferably, the anti-inflammatory factor is selected from interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-13 (IL-13) and Two or more of interleukin-37 (IL-37), preferably interleukin 10 and interleukin 37.
  • IL-3 interleukin-3
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • IL-6 interleukin-6
  • IL-10 interleukin-10
  • IL-13 interleukin-13
  • IL-37 Two or more of interleukin-37
  • the nucleic acid encoding interleukin 10 has the amino acid sequence shown in SEQ ID NO: 1
  • the nucleic acid encoding interleukin 37 has the amino acid sequence shown in SEQ ID NO: 2.
  • Interleukin 10 a pleiotropic cytokine
  • IL-10 can play an immunosuppressive or immunostimulatory role in various types of cells.
  • MHC II major histocompatibility complex antigen II
  • IL-10 can also inhibit the synthesis and release of inflammatory factors and play an anti-inflammatory role.
  • IL-10 can inhibit the production of prostaglandin E2 and inflammatory cytokines including TNF- ⁇ , IL-1, IL-6 and IL-8.
  • IL-10 can inhibit the proliferation of human THO, Th-1, Th-2-like T cell clones. Promote the proliferation of mast cells and thymocytes, and IL-10 is also a complex factor for the growth of lymph nodes and spleen cells.
  • IL-10 suppresses inflammatory and cellular immune responses, enhances tolerance associated with adaptive immunity and clearance, and suppresses proinflammatory cytokines produced by monocytes and macrophages.
  • Interleukin 37 is a newly discovered IL-1 family cytokine, which can inhibit both innate immunity and adaptive immunity. IL-37 is mainly expressed in neutrophils, lymphocytes, macrophages, monocytes, tissue epithelial cells, keratinocytes and dendritic cells. Under the stimulation of a variety of Toll-like receptors and inflammatory cytokines such as IL-1B, tumor necrosis factor (TNF) ⁇ and interferon ⁇ , IL-37 in peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC), epithelial cells , macrophages and DCs were induced.
  • TNF tumor necrosis factor
  • the present inventors found that the combination of the nucleic acid encoding interleukin 10 and the nucleic acid encoding interleukin 37 can effectively inhibit the innate immune response, effectively regulate the adaptive immune response, and maintain the immune tolerance of the body. Immune cells are activated, thereby improving the body's own inflammatory environment. .
  • the nucleic acid encoding interleukin 10 and the nucleic acid encoding interleukin 37 are contained in the same or different expression vectors, preferably contained in the same expression vector.
  • nucleic acid encoding interleukin 10 and the nucleic acid encoding interleukin 37 are connected by a sequence encoding a self-cleaving peptide, for example, a nucleic acid having a sequence as shown in SEQ ID NO: 3, which encodes A T2A polypeptide having an amino acid sequence as shown in SEQ ID NO:3.
  • sequence encoding the self-cleaving peptide is linked between the 3' end of the nucleic acid encoding interleukin 10 and the 5' end of the nucleic acid encoding interleukin 37.
  • the nucleic acid encoding an anti-inflammatory factor is carried by a strong promoter that is easy to be silenced in stem cells, the main purpose is to make it secrete a large amount of inflammatory inhibitory factors in the early stage of stem cell transplantation so as to improve the quality of the body.
  • CMV is preferably used, which can efficiently and quickly drive and induce the expression of anti-inflammatory factors, quickly form an inflammatory suppression environment, and provide a good therapeutic environment.
  • the strong promoter is linked to the 5' end of the nucleic acid encoding interleukin-37.
  • the cartilage repair factor is selected from fibroblast growth factors such as FGF2a, FGF2b, FGF18 or FGF23, etc., more preferably, the cartilage repair factor is selected from Fibroblast growth factor 18 (FGF18).
  • FGF18 Fibroblast growth factor 18
  • the nucleic acid encoding fibroblast growth factor 18 has the amino acid sequence shown in SEQ ID NO: 4.
  • Fibroblast growth factors are polypeptides consisting of approximately 150-200 amino acids that exist in two closely related forms, basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF). Both FGFs stimulate DNA synthesis in isolated rat calvarial osteoblast-like cells, while bFGF stimulates colony formation of differentiated chondrocytes in agar, acting as a mitogen and morphogen. FGF increases the number of poorly differentiated preosteoblasts in vivo. Adding FGF to chicken embryos can make osteoblasts and chondrocytes proliferate and form bone matrix.
  • bFGF basic fibroblast growth factor
  • aFGF acidic fibroblast growth factor
  • FGF18 is a highly conserved protein consisting of 207 amino acids, which has 30%-70% homology with other members of the discovered FGFs. FGF18 protein plays an important role in bone growth and development, and is also involved in cortical neuron activity, adenohypophysis survival, differentiation and proliferation, and regulation of hair growth and skin repair.
  • the directional differentiation of stem cells is different according to the microenvironment, and the existence of an inflammatory environment is not conducive to the differentiation of stem cells towards bone and cartilage.
  • the inventors of the present invention have discovered that by allowing stem cells to continuously secrete anti-inflammatory factors such as IL10 and IL37, improving their own differentiation microenvironment, continuously alleviating local injection inflammatory reactions, and then combining with the factor (FGF18) that drives stem cells to differentiate into cartilage, the stem cells can be better.
  • the nucleic acid encoding the cartilage repair factor is carried by a medium-strength promoter that is not easy to be silenced in stem cells, which helps to stably express the cartilage repair factor in the initially rapidly formed inflammatory suppression environment , to obtain a stable cartilage repair effect.
  • EF1 ⁇ EF1 ⁇ , SV40, PGK1 or Ubc, etc.
  • EF1 ⁇ promoter a strong mammalian cell expression promoter derived from human elongation factor 1 ⁇ , its expression level is very stable, and it is compatible with cell types and Where the cell is located makes it irrelevant.
  • the medium-strength promoter is preferably connected to the 5' end of the nucleic acid encoding a cartilage repair factor through an enhancer.
  • the genetically modified stem cells further include an expression vector, and the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor are contained in the same or different expression vectors, preferably contained in the same expression vector. in the carrier.
  • nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor are connected through a medium-strength promoter.
  • the nucleic acid encoding the anti-inflammatory factor is linked to the nucleic acid encoding the cartilage repair factor, for example, through a medium-strength promoter.
  • a medium-strength promoter such as EF1a is linked to an enhancer, the enhancer is linked to the 5' end of the nucleic acid encoding a cartilage repair factor, and the medium-strength promoter is linked to a nucleic acid encoding an anti-inflammatory factor.
  • the stem cells are mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow or umbilical cord blood, preferably from umbilical cord.
  • the mesenchymal stem cell is a kind of pluripotent stem cell, which has all the common characteristics of stem cells, that is, self-renewal and multidirectional differentiation capabilities. In addition, it has the function of immune regulation, through the interaction between cells and the production of cytokines to inhibit the proliferation of T cells and their immune response, thereby exerting the function of immune reconstruction; at the same time, the source of mesenchymal stem cells is convenient, easy to separate, culture, expand Increased and purified, after multiple passages and expansion, it still has the characteristics of stem cells and does not have the characteristics of immune rejection.
  • Umbilical cord mesenchymal stem cells are derived from isolated umbilical cord tissue, which is convenient to obtain materials from a wide range of sources and is not subject to ethical debates and restrictions. Rapid growth, low immunogenicity, no tumorigenicity, can be used as a cell carrier for gene therapy. Under specific induction conditions in vivo or in vitro, umbilical cord mesenchymal stem cells can differentiate into a variety of tissue cells, and they still have multidirectional differentiation potential after continuous subculture and cryopreservation. They are ideal seed cells for cell transplantation therapy.
  • umbilical cord-derived mesenchymal stem cells are preferably used in the present invention.
  • the invention provides a method for preparing gene-modified stem cells used for cartilage repair therapy, comprising linking nucleic acids encoding anti-inflammatory factors and nucleic acids encoding cartilage repair factors, and introducing them into mesenchymal stem cells.
  • the preparation method comprises the following steps:
  • Step 1 obtaining the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor, digesting the nucleic acid and connecting with the carrier plasmid to obtain the recombinant plasmid;
  • Step 2 transfecting the recombinant plasmid and the packaging plasmid into lentiviral packaging cells to obtain a recombinant lentiviral vector containing nucleic acids encoding anti-inflammatory factors and nucleic acids encoding cartilage repair factors;
  • Step 3 transfecting the recombinant lentiviral vector obtained above into mesenchymal stem cells to obtain genetically modified stem cells for cartilage repair therapy.
  • the preparation method of the genetically modified stem cells is further described as follows:
  • Step 1 Obtain the nucleic acid encoding the anti-inflammatory factor and the nucleic acid encoding the cartilage repair factor, digest with the vector plasmid, and obtain the recombinant plasmid.
  • the nucleic acid encoding an anti-inflammatory factor is a nucleic acid encoding a double anti-inflammatory factor, which can simultaneously encode IL10 and IL37, and the part encoding IL10 has a nucleotide sequence as shown in SEQ ID NO: 1; its encoding Part of IL37 has the nucleotide sequence shown in SEQ ID NO:2.
  • the nucleic acid encoding IL10 and the nucleic acid encoding IL37 are preferably connected by a T2A sequence, and the T2A sequence is shown in SEQ ID NO:3.
  • the part encoding IL10 in the nucleic acid encoding double anti-inflammatory factor is connected with strong promoter CMV.
  • the nucleic acid encoding the cartilage repair factor is the nucleic acid encoding FGF18, which has a nucleotide sequence as shown in SEQ ID NO:4.
  • the 5' end of the nucleic acid encoding FGF18 is connected to a medium-strength promoter through an enhancer 5'LTR sequence, such as the EF1a sequence having the nucleotide sequence shown in SEQ ID NO:7.
  • the part encoding IL37 in the nucleic acid encoding the anti-inflammatory factor is connected with the enhancer 5'LTR sequence (it has the nucleotide sequence shown in SEQ ID NO: 5) through the promoter EF-1 ⁇ , the 5'LTR sequence is then connected to the nucleic acid encoding the cartilage repair factor.
  • the methods commonly used in the prior art are used to digest the nucleic acid part encoding the nucleic acid part of the anti-inflammatory molecule and the nucleic acid part encoding the cartilage repair factor, and carry out enzyme digestion on the vector plasmid, and perform 4-year reaction with T4 ligase.
  • transform DH5 ⁇ competent cells Connect overnight at °C, transform DH5 ⁇ competent cells, take 100 ⁇ L of the bacterial solution and spread it on an LB plate containing ampicillin resistance, culture overnight at 37°C, pick a single clone for colony PCR, send the positive clone for sequencing, and save for sequencing
  • the plasmid was correctly cloned and extracted, and its map is shown in Figure 1.
  • the carrier plasmid is pCDH-CMV
  • the recombinant plasmid is pCDH-103718
  • the enzymes used for digestion are preferably XbaI and SaII.
  • Step 2 Transfect the recombinant plasmid and packaging plasmid into lentiviral packaging cells to obtain a recombinant lentiviral vector containing nucleic acids encoding anti-inflammatory molecules and nucleic acids encoding cartilage repair molecules.
  • step 2 includes the following sub-steps:
  • Step 2-1 culturing packaging cells
  • the packaging cells are preferably 293T cells.
  • Activated 293T cells are resuspended, inoculated into a culture dish for culture, and digested when the cell confluence reaches 90% or more; after the digestion is terminated, the cells are centrifuged , resuspended, and inoculate cells in each coated culture dish for packaging virus, preferably each culture dish (150mm) inoculates 1 ⁇ 10 7 ⁇ 1.5 ⁇ 10 7 cells, preferably each culture dish inoculates 1.2 ⁇ 10 7 cells.
  • the culture medium of 293T cells is 10% FBS+1mM sodium pyruvate+2mM glutamine+1% non-essential amino acid+DMEM medium.
  • Step 2-2 transfect packaging cells
  • the packaging plasmid was mixed with the recombinant cloning plasmid (pCDH-103718) obtained in step 1, and after incubation, a transfection reagent was added and incubated at room temperature to form a DNA-transfection reagent complex.
  • the packaging plasmid can be a lentiviral packaging helper plasmid commonly used in the prior art, such as PMD.2G and PSPAX purchased from Addgene, preferably the mass ratio of the PMD.2G plasmid and the PSPAX plasmid is 2:1, More preferably, the mass ratio of the PMD.2G plasmid, the PSPAX plasmid and the pCDH-103718 plasmid is 2:1:3.
  • PMD.2G and PSPAX purchased from Addgene
  • the transfection reagent can be a reagent commonly used in the prior art, for example, PEI (purchased from Polyscience) solution, and the transfection reagent is mixed with DMEM medium and added in drops to the above plasmid mixing system.
  • PEI purchased from Polyscience
  • the DNA-transfection reagent complex was mixed evenly with the packaging cells, cultured, the medium was changed regularly during the culture process, and the supernatant liquid was collected and stored at 4°C after the medium change.
  • the collected supernatant liquid is concentrated, and the virus titer is determined
  • the concentration is preferably low-temperature centrifugation of the supernatant liquid.
  • Step 3 transfecting the recombinant lentiviral vector obtained above into mesenchymal stem cells to obtain genetically modified stem cells.
  • the stem cells are mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow or umbilical cord blood, preferably from umbilical cord.
  • the mesenchymal stem cells are isolated by the umbilical cord tissue block slide method.
  • the isolated umbilical cord of normal delivery into PBS buffer solution containing 200 U/mL penicillin and 200 U/mL streptomycin.
  • the fresh umbilical cord should be separated within 6 hours.
  • Flush the residual blood in the umbilical vein and umbilical artery with a 20mL syringe cut the umbilical cord tissue into 1mm3 tissue pieces with tissue scissors, then filter the obtained small pieces of umbilical cord tissue with a 200-mesh filter, and collect 200-mesh
  • the umbilical cord tissue blocks on the strainer remove the too small umbilical cord tissue blocks, and obtain multiple umbilical cord tissue blocks with a diameter of 1-1.5 mm.
  • Tissue blocks with a diameter of 1-1.5 mm were collected, inoculated directly into culture bottles, placed in a 5% CO 2 , 37°C incubator, and left to stand for 1-2 hours. After the tissue block is firmly attached to the wall, add ⁇ -MEM culture solution containing 10% fetal bovine serum, and place it in a 5% CO 2 , 37°C incubator to continue culturing. Five days later, the umbilical cord tissue mesenchyme in the culture bottle The proliferation of stem cells is about 80% confluent; digested with 0.25% trypsin (0.01% EDTA), the obtained cells are primary cells. MSCs were isolated and cultured by the umbilical cord tissue slice method. After 72 hours, a small amount of cells crawled out around the umbilical cord tissue. After about 7 days, the cells dissociated from the tissue and gradually formed clones. The obtained mesenchymal stem cells were cryopreserved.
  • Step 3 includes the following sub-steps:
  • Step 3-1 culturing and inoculating stem cells
  • the pre-frozen stem cells were resuscitated and cultured. After the resuscitated cells were overgrown, the cells were digested with 0.05% trypsin, and the digestion was terminated with a serum-containing medium. After the digestion was terminated, the cells were centrifuged and resuspended. Then inoculate the cells into a petri dish, preferably 2 ⁇ 10 6 -2.5 ⁇ 10 6 cells per petri dish (150 mm), and replace the medium the next day after inoculation.
  • Step 3-2 add lentiviral vector and continue culturing
  • Polybrene Polybrene
  • the volume of lentiviral vector added (MOI ⁇ number of cells)/virus titer.
  • the MOI refers to the ratio of the number of viruses to cells during transduction.
  • the MOI is preferably 10-50, such as 40.
  • Step 3-3 subculture the cells
  • the cells to be genetically modified are overgrown, the cells are digested with 0.05% trypsin, the digestion is terminated with a medium containing serum, the cell suspension is centrifuged at 800rpm for 5min, and the cells obtained by centrifugation are resuspended with a serum-free medium, according to The subculture ratio was 1:6, cultured in serum-free medium at 37°C and 5% CO 2 for 3 days until the cells became confluent, and the genetically modified stem cells were obtained, which were designated as MSC-103718.
  • the present invention also provides the genetically modified stem cells prepared by the above method.
  • the present invention also provides the use of the above-mentioned genetically modified stem cells in the preparation of medicines for the repair/treatment of cartilage diseases.
  • cartilage diseases include acute cartilage injury, chronic articular cartilage wear, relapsing polychondritis, etc., preferably chronic articular cartilage wear.
  • cartilage diseases manifest as degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, hyperosteogeny, rheumatoid arthritis, and rheumatoid arthritis.
  • the present invention also provides a pharmaceutical composition, which includes the above-mentioned genetically modified stem cells or the stem cells prepared by the above-mentioned method.
  • the dosage form of the pharmaceutical composition can be any form known in the medical field, preferably tablet, pill, suspension, emulsion, solution, injection, gel, capsule, powder, granule or suppository, Injections are more preferred.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient, preferably a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (for example: balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • a pharmaceutically acceptable carrier or excipient preferably a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (for example: balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the pharmaceutical composition can be implanted in the form of suspension, gel, colloid, slurry or mixture.
  • the configuration of the pharmaceutical composition is carried out according to the principle of cell medicine prescribed in the prior art, for example, it can be carried out according to "Hematopoietic Stem Cell Therapy", E.D.Ball, J.Lister&P.Law, Churchill Livingstone, 2000 .
  • the pharmaceutical composition can be administered to the subject by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection or oral administration,
  • the subject is a mammal, such as a human.
  • IL10 encoding nucleotide (nucleotide sequence is shown in SEQ ID NO: 1) and IL37 encoding nucleotide (nucleotide sequence is shown in SEQ ID NO: 2) with T2A (nucleotide sequence is shown in SEQ ID NO : 3) connection, FGF18 encoding nucleotide (nucleotide sequence shown in SEQ ID NO: 4) before connecting 5'LTR sequence (sequence shown in SEQ ID NO: 5) and EF1 ⁇ promoter (sequence shown in SEQ ID NO: 5) and EF1 ⁇ promoter (sequence shown in SEQ ID NO: 4) ID NO: 7), IL37 was connected to the EF1 ⁇ promoter, restriction sites XbaI and SaII were introduced at both ends, and human XbaI-IL10-T2A-IL37-EF1 ⁇ -5'LTR-FGF18-SaIl was synthesized, named as Sequence1037
  • the pCDH-CMV plasmid (Addgene) was digested with XbaI and SaII, and a fragment of about 6195 bp was recovered from the product gel as a vector.
  • Sequence103718 was digested with XbaI and SaII, and a fragment of about 2487bp was recovered from the product gel.
  • 293T cell preparation Take out a frozen 293T cell (purchased from ATCC) from liquid nitrogen and quickly put it in a 37°C water bath until the ice disappears, then add it dropwise to a 15ml centrifuge tube containing 5ml preheated medium , centrifuge at 1200rpm for 3min, discard the supernatant, resuspend the cells in 293T medium (10% FBS+1mM sodium pyruvate+2mM glutamine+1% non-essential amino acid+DMEM) and inoculate them in a 150mm culture dish at 37°C for 5 Incubation in % CO 2 saturated humidity.
  • 293T medium (10% FBS+1mM sodium pyruvate+2mM glutamine+1% non-essential amino acid+DMEM
  • the mesenchymal stem cells were isolated by the umbilical cord tissue block slide method, and the specific steps were as follows:
  • the fresh umbilical cord should be separated within 6 hours.
  • Flush the residual blood in the umbilical vein and umbilical artery with a 20mL syringe cut the umbilical cord tissue into 1mm3 tissue pieces with tissue scissors, then filter the obtained small pieces of umbilical cord tissue with a 200-mesh filter, and collect the 200-mesh filter
  • Umbilical cord tissue blocks on the net remove too small umbilical cord tissue blocks, and obtain multiple umbilical cord tissue blocks with a diameter of 1-1.5 mm.
  • Tissue blocks with a diameter of 1-1.5 mm were collected, inoculated directly into culture bottles, placed in a 5% CO 2 , 37°C incubator, and left to stand for 1-2 hours. After the tissue block is firmly attached to the wall, add ⁇ -MEM culture solution (purchased from Gibco) containing 10% fetal bovine serum, and place it in a 5% CO 2 , 37°C incubator to continue culturing. Five days later, in a culture bottle The proliferation of umbilical cord tissue mesenchymal stem cells is about 80% confluent; digested with 0.25% trypsin (0.01% EDTA), the obtained cells are primary cells. MSCs were isolated and cultured by the umbilical cord tissue slice method. After 72 hours, a small amount of cells crawled out around the umbilical cord tissue. After about 7 days, the cells dissociated from the tissue and gradually formed clones. The obtained mesenchymal stem cells were cryopreserved.
  • Each 150mm culture dish was inoculated with 2-2.5 ⁇ 106 cells, the culture medium of the cells was sucked out the next day after inoculation, and replaced with serum-free ⁇ -MEM medium, 20ml medium/dish, and 16 ⁇ l Polybrene (purchased) was added.
  • serum-free ⁇ -MEM medium 20ml medium/dish, and 16 ⁇ l Polybrene (purchased) was added.
  • the LV-103718 lentivirus obtained in Example 4 (with a titer of 1 ⁇ 10 8 U/ml) was added at the same time according to the multiplicity of infection of 40 MOIs, and cultured at 37° C., 5% CO 2 saturated humidity for 6-8 hours. Discard the ⁇ -MEM medium containing the virus after 6-8 hours, replace it with a serum-free medium, and continue to cultivate for 2-3 days at 37° C.
  • the LV-103718 gene-modified MSCs can highly express IL10, IL37 and FGF18 factors.
  • Inoculate hMSCs with maintenance medium pre-coat 24-well plates with MSCAttachmentsolution (BI; P/N:05-752-1, diluted 1:100 in DPBS), add 0.5ml MSC NutriStem to each well XF, inoculate 6x104 cells (3x104cells/cm2). Place in a 37°C, 5% CO 2 cell culture incubator.
  • Induce differentiation with adipogenic differentiation medium after 24 hours of culture, confirm that the degree of cell confluence has reached 80-90%, remove the maintenance medium, and add 0.5 ml of differentiation medium to each well (24-well plate). Place in a 37°C, 5% CO 2 cell culture incubator for 14-21 days. The operation of changing the medium during this period is as follows, use the complete differentiation medium to culture for 6-8 days, and change the medium every 3-4 days during this period. After differentiation is complete, replace the medium with maintenance medium. Mature adipocytes (i.e. lipid droplet formation) are observed and can be stained.
  • Mature adipocytes i.e. lipid droplet formation
  • Oilred-O staining process Aspirate the medium and wash once with DPBS (1ml/well, 24-well plate). Fixation: DPBS was removed by suction, and 10% formalin (4% formaldehyde; 1ml/well, 24-well plate) was added. Fix at room temperature for 30-60 minutes. The formalin was removed by suction, and washed with 60% isopropanol for 2-3 minutes (1 ml/well, 24-well plate). Aspirate the isopropanol, add Oilred-O staining working solution (1ml/well, 24-well plate). Let stand at room temperature for 10-30 minutes. Wash with distilled water to remove excess dye. The staining effect is shown in Figure 4, where Sample 1 and Sample 2 are two independent samples from different individuals.
  • Inoculation of hMSCs pre-coat 24-well plates with MSCAttachmentsolution (BI; P/N:05-752-1, diluted 1:100 in DPBS), add 0.5ml MSC NutriStem to each well XF, inoculate 6x10 4 cells (3x10 4 cells/cm 2 ). Place in a 37°C, 5% CO 2 cell culture incubator.
  • Induce differentiation with osteogenic differentiation medium After 24 hours of culture, confirm that the degree of cell confluence has reached 80-90%, remove the maintenance medium, and add 0.5 ml of differentiation medium to each well (24-well plate). Place in a 37°C, 5% CO 2 incubator for 10-21 days, and change the medium every 2-3 days.
  • Osteogenesis evaluation Aspirate the culture medium, wash once with DPBS (BI; 02-023-1) (1ml/well). Fixation: Remove DPBS by suction and add 1ml of 70% EtOH to each well. Fix at room temperature for 30-60 minutes. EtOH was sucked off and washed 3 times with distilled water (1ml/well). Aspirate the distilled water, add 1ml 2% ARS staining working solution to each well. Let stand at room temperature for 30-60 minutes. Aspirate the staining solution and wash 4 times with 1ml distilled water (1ml/well). Add 1ml of distilled water to each well to avoid drying the cells. The effect of osteogenic staining is shown in Figure 5, where Sample 1 and Sample 2 are two independent samples from different individuals.
  • Inoculate hMSCs with maintenance medium cells were inoculated at 1x10 5 /(10 ⁇ l medium) into a 1-well U-bottom 96-well plate (non-TC-treated) and placed in a 5% CO 2 cell culture incubator at 37°C for 2 hours (promoted cells aggregated into clumps), carefully add 0.1ml of medium.
  • the culture method of small microaggregates is conducive to the formation of spherical cell clusters, put them back into a 37°C, 5% CO 2 cell incubator to continue culturing.
  • Induce differentiation with chondrogenic differentiation medium after 24 hours of culture, remove the maintenance medium, add 0.2ml of differentiation medium to each well (96-well plate), and spherical cell clusters will form in 24-48 hours. Place in a 37°C, 5% CO 2 cell culture incubator for 14-21 days. Change the differentiation medium every 3-4 days. Longer culture time helps to obtain more mature chondrocytes.
  • AlcianBlue staining process Aspirate the medium and wash once with DPBS (BI; 02-023-1, 0.2ml/well). Fixation: DPBS was removed by suction, and 0.2 ml of 10% formalin (4% formaldehyde) was added to each well. Fix at room temperature for 30-60 minutes. The formalin was removed by suction, and each well was washed twice with 0.2ml distilled water. Aspirate the distilled water, and add 0.2ml of AlcianBlue staining working solution to each well. overnight at room temperature in the dark. Aspirate the dye solution, wash each well with 0.2ml 0.1N HCl 2-3 times. HCl was removed by suction, and 0.2 ml of distilled water was added to each well. The staining results are shown in Figure 6 and Figure 7, where Sample 1-Sample 4 are four independent samples from different individuals.
  • stem cells MSC-103718 modified by LV-103718 can rapidly differentiate into osteoblasts and chondrocytes, and the efficiency is significantly higher than that of unmodified MSC cells; LV-103718 The -103718 modification had no significant effect on adipogenic differentiation.
  • PBMC isolation and culture Transfer 10ml of whole blood into a 50ml centrifuge tube, add 10ml of PBS solution to dilute, and mix gently; take two 15ml centrifuge tubes, first add 5ml of lymphocyte separation (ficoll) solution. Then gently add the diluted blood to the upper layer of ficoll of the two centrifuge tubes, be sure to be gentle to avoid mixing the two solutions together, each centrifuge tube has 10ml of diluted blood; 2,000rpm, 20min, note that the deceleration setting must be It should be set to nobreak, or only 1-2% of the brake. Centrifugation is completed; the cell layer where PBMCs are located is white.
  • the layer of cells can be sucked into another clean 15ml centrifuge tube with a pipette.
  • MSCs (MSC group, MSC-1037 group, and MSC-18 group) are co-cultured with PBMCs: each group of MSCs (MSC group, MSC-1037) in a good growth state (obtained by the method of embodiment 1-4, the difference is Synthesized human source XbaI-IL10-T2A-IL37-EF1 ⁇ -Sall ) group, MSC-18 (obtained with the method of embodiment 1-4 in embodiment 1, the difference is that synthetic human source XbaI-EF1 ⁇ -5' in embodiment 1 LTR-FGF18-Sall ) group and MSC-103718 group) were counted, centrifuged and resuspended to adjust the cell concentration to 3 ⁇ 10 5 /ml, each was inserted into a 6-well plate at 2ml/well, and 3 wells were paralleled, 37°C, 5 After 16-18 hours of culture with %CO2, the cells were stably adhered to the wall, and then the medium was changed, mixed according to the ratio
  • MSC-103718 group can better inhibit the proliferation of total lymphocytes, inhibit the proliferation of Th1 and Th17 lymphocytes, and promote the proliferation of Treg cells; as shown in Figure 9: Compared with other groups, MSC-103718 group can better inhibit the expression of TNF- ⁇ cytokine.
  • Cartilage repair ability to cartilage directional differentiation culture when P1 subcultured cells are cultured until the cells cover the bottom of the bottle, adjust the cell concentration after digestion, add high-sugar DMEM solution containing TGF ⁇ 1 (10ng/ml) and 10% fetal bovine serum to induce nourish.
  • the cell suspensions of different groups of MSC group, MSC-1037 group, MSC-18 group and MSC-103718 group were adjusted in concentration, dropped onto the PLGA support, and cultured in a 37°C, 5% CO 2 incubator for 7-d.
  • the complex was transplanted into 10 healthy Beagle dogs aged 10 to 12 months. After successful intravenous anesthesia and preparation of the operation area, an incision was made on the inside of the knee joint to expose the knee joint. Cylindrical cartilage defect, mm in diameter, deep to the subchondral bone. Gross observations were taken at 12 and 16 weeks after operation to observe the growth of cartilage in the defect area.
  • the tissue in the defect repair area of the MSC-103718 group was integrated with the surrounding articular cartilage, and the cartilage defect area was covered by smooth white translucent tissue, which had no difference in shape from the surrounding cartilage tissue; the defect area of the MSC-Con and MSC-1037 groups The repaired tissue was partly integrated with the surrounding cartilage, and the luster was poor; the repaired tissue in the defect of MSC-18 group was soft and dull, and the new tissue was soft and dull, which was obviously different from the surrounding cartilage tissue.
  • SEQ ID NO:1 encodes the nucleotide sequence (534bp) of IL10
  • SEQ ID NO:6 The nucleotide sequence (2506bp) of XbaI-IL10-T2A-IL37-EF-1 ⁇ -5'LTR-FGF18-SaIl

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Abstract

提供了一种用于软骨修复治疗的基因修饰的干细胞及其制备方法和用途，还提供了包括该干细胞的药物组合物。其中，所述干细胞包括编码抗炎因子的核酸和编码软骨修复因子的核酸，通过采用抗炎因子和与促进软骨分化的因子同时修饰间充质干细胞，不仅能有效促进间充质干细胞分化为软骨，而且通过多种因子的结合全面作用于各类免疫细胞，抑制各类免疫细胞活性，从而解决骨膝等部位软骨修复的局部炎性环境问题，使得临床患者在应用间充质干细胞进行骨膝修复时降低或减少肿胀情况发生。

Description

一种基因修饰干细胞及其用途 技术领域
本发明涉及基因工程，尤其涉及干细胞治疗技术领域，具体涉及用于软骨修复的基因修饰干细胞。
背景技术
干细胞是一类具有自我更新和分化潜能的细胞，是肿瘤和心血管疾病及其他恶性疾病研究或治疗的重要手段。干细胞治疗在生命科学、新药试验和疾病研究这三大领域中具有巨大研究和应用价值，现已广泛应用于医药再生细胞替代治疗和药物筛选等领域，成为世界关注和研究的焦点。
干细胞药品涉及的适应症主要是膝骨关节炎和糖尿病足多种。干细胞临床治疗研究疾病涉及全身各大系统，涉及疾病包括：急性心梗、小儿脑性瘫痪、卵巢早衰、银屑病、间质性肺病、膝骨关节炎、帕金森病、视网膜色素变性、年龄相关性黄斑变性、溃疡性结肠炎、骨修复、空鼻综合症、不孕症、狼疮性肾炎、视神经脊髓炎、薄性子宫内膜、COPD所致肺动脉高压、失代偿性乙型肝炎肝硬化、神经病理性疼痛、半月板损伤等。
用于干细胞治疗的干细胞种类包括：胚胎干细胞衍生细胞、神经干细胞、各种来源的间充质干细胞(如脂肪、脐带、骨髓、胎盘等)、支气管基底层细胞。涉及的治疗方式包括：细胞单独使用，细胞联合材料使用，细胞联合药物使用。涉及的细胞来源：自体和异体。
目前虽然对干细胞治疗骨膝关节修复等临床疾病均具有极大的需求和支持，不过，由于目前对间充质干细胞调节其活性的因素理解有限，以及缺乏对间充质干细胞和它们的生态微环境的组分之间的复杂相互作用的了解，间充质干细胞临床应用的有效性和安全性受到限制，因此致使间充质干细胞修复骨膝关节的相关研究仍有很大的探索空间。
发明内容
为了克服上述问题，本发明人进行了锐意研究，结果发现：采用抗炎因子和与促进软骨分化的因子同时修饰间充质干细胞，不仅能有效促进间充质干细胞分化为软骨，而且通过多种因子的结合全面作用于各类免疫细胞，抑制各类免疫细胞活性，从而解决骨膝等部位软骨修复的局部炎性环境问题，使得临床患者在应用间充质干细胞进行骨膝修复时降低或减少肿胀情况发生，从而完成了本发明。
具体地，本发明提供以下方面：
第一方面，提供一种用于软骨修复治疗的基因修饰干细胞，所述干细胞包括编码抗炎因子的核酸和编码软骨修复因子的核酸。
第二方面，提供如上所述用于软骨修复治疗的基因修饰干细胞，其中所述抗炎因子选自白细胞介素和TGFβ受体家族，优选地，所述抗炎因子选自白细胞介素-3(IL-3)、白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-13(IL-13)和白介素-37(IL-37))中的两种或更多种，更优选为白细胞介素10和白细胞介素37。
第三方面，提供如上所述用于软骨修复治疗的基因修饰干细胞，其中所述软骨修复因子选自成纤维细胞生长因子、FGF2a
、FGF2b、FGF18、FGF23，优选地，所述软骨修复因子选自成纤维细胞生长因子18(FGF18)。
第四方面，提供如上所述用于软骨修复治疗的基因修饰干细胞，所述编码抗炎因子的核酸通过易在干细胞中沉默的强启动子携带，所述编码软骨修复因子的核酸采用不易在干细胞中沉默的中等强度启动子携带。
第五方面，提供如上所述用于软骨修复治疗的基因修饰干细胞，编码抗炎因子的核酸和编码软骨修复因子的核酸连接。
第六方面，提供用于软骨修复治疗的基因修饰干细胞的制备方法，包括将编码抗炎因子的核酸和编码软骨修复因子的核酸连接，并导入间充质干细胞。
第七方面，提供如上所述用于软骨修复治疗的基因修饰干细胞的制备方法，包括以下步骤：
步骤1，获得编码抗炎因子的核酸和编码软骨修复因子的核酸，酶切后与载体质粒连接，得到重组质粒；
步骤2，将重组质粒与包装质粒进行慢病毒包装细胞的转染，得到含有编码抗炎因子的核酸和编码软骨修复因子的核酸的重组慢病毒载体；
步骤3，将上述获得的重组慢病毒载体转染至间充质干细胞，得到用于软骨修复治疗的基因修饰的干细胞。
第八方面，提供通过上述方法制备得到的基因修饰的干细胞。
第九方面，提供一种上述基因修饰干细胞或上述方法制备得到的基因修饰的干细胞在制备用于软骨性疾病的修复/治疗药物中的用途。
第十方面，提供如上所述的用途，其中软骨性疾病包括退行性关节炎、滑囊炎、滑膜炎、颈椎病、腰椎病、肩周炎、骨质增生、风湿性关节炎和类风湿性关节炎等。
本发明所具有的有益效果包括：
(1)本发明提供的基因修饰的干细胞，能够同时表达抗炎因子和软骨修复因子，具有良好的协同效应，不仅能有效促进间充质干细胞分化为软骨，而且能抑制各类免疫细胞活性，降低或避免软骨修复时局部炎性；
(2)本发明提供的基因修饰的干细胞，既保持间充质干细胞的功能，又能很好的表达脂抗炎因子和软骨修复因子，可安全地施用给人受试者，而不引发免疫原性反应；
(3)本发明提供的基因修饰的干细胞，具备显著改善患者膝部等软骨修复时的炎性，有效缓解应用间充质干细胞在进行骨膝修复时减少/避免肿胀等问题，具有重大的临床价值。
附图说明
图1示出实施例的pCDH-103718质粒质粒图谱；
图2示出实验例中MSC-103718细胞的表型结果图；
图3示出实验例中MSC-103718细胞中IL10、IL37和FGF18的表达量的检测结果示意图；
图4示出实验例中各样品诱导分化培养21天成脂分化能力检测结果示意图；
图5示出实验例中各样品诱导分化培养21天成骨分化能力检测结果示意图；
图6示出实验例中各样品诱导分化培养21天成软骨分化能力检测结果放大示意图；
图7示出实验例中各样品诱导分化培养21天成软骨分化能力检测结果示意图；
图8示出MSC-103718细胞对免疫细胞的调控作用结果示意图；
图9示出MSC-103718细胞对淋巴细胞分泌TNF-α抑制结果示意图。
具体实施方式
下面通过优选实施方式和实施例对本发明进一步详细说明。通过这些说明，本发明的特点和优点将变得更为清楚明确。
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
一、关于基因修复的间充质干细胞
本发明人通过抗编码炎因子的核酸和编码软骨修复因子的核酸共同修饰间充质干细胞，展现出多基因显著的协同效应，具有重大的临床价值。
根据本发明提供的基因修饰的干细胞，抗编码炎因子的核酸和编码软骨修复因子的核酸。
在一个优选的实施方式中，所述抗炎因子选自白细胞介素和TGFβ受体家族，优选地，所述抗炎因子选自白细胞介素-3(IL-3)、白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-13(IL-13)和白细胞介素-37(IL-37))中的两种或更多种，优选为白细胞介素10和白细胞介素37。
优选地，编码白细胞介素10的核酸具有SEQ ID NO：1所示的氨基酸序列，编码白细胞介素37的核酸具有SEQ ID NO：2所示的氨基酸序列。
白细胞介素10(IL-10)，是一种多效性细胞因子，可以在多种类型细胞中发挥免疫抑制或免疫刺激的作用。在炎症反应方面，能通过下调单核细胞表面主要组织相容性抗原Ⅱ(classⅡmajorhistocompatibilitycomplex,MHCⅡ)的表达，降低其抗原呈递作用，下调T淋巴细胞活性,抑制炎性细胞的激活、迁移和粘附；同时，IL-10也能抑制炎症因子的合成与释放，起到抗炎的作用。作为细胞介导免疫反应的负调节物，IL-10能抑制前列腺素E2和炎性细胞因子包括TNF-α、IL-1、IL-6和IL-8的产生。IL-10可抑制人THO、Th-1、Th-2样T细胞克隆的增殖。促进肥大细胞和胸腺细胞增殖，IL-10也是淋巴结、脾脏细胞生长的复合因子。IL-10抑制炎症反应和细胞免疫反应，加强与适应性免疫和清除功能相关的耐受性，抑制由单核细胞和巨噬细胞产生的促炎因子。提高B细胞的存活率，促进B细胞的增殖、MHCⅡ类抗原表达以及免疫球蛋白的分泌，并与Th2所产生的IL-4、IL-5有协同作用。抑制NK细胞因子的产生。抑制氮氧化物的产生。
白细胞介素37(IL-37)是新近发现的IL-1家族细胞因子，对固有免疫和适应性免疫均有抑制作用。IL-37主要表达于中性粒细胞、淋巴细胞、巨噬细胞、单核细胞、组织上皮细胞、角质形成细胞和树突状细胞。在多种Toll样受体和炎性细胞因子如IL-1B、肿瘤坏死因子(TNF)α以及γ干扰素等的刺激下，IL-37在外周血单个核细胞(peripheralbloodmonocytecell，PBMC)、上皮细胞、巨噬细胞以及DC等诱导产生。
本发明人发现，编码白细胞介素10的核酸和编码白细胞介素37的核酸组合能够有效抑制固有免疫应答，有效调节适应性免疫应答，维持机体免疫耐受，两者结合更能够全面抑制各类免疫细胞活化，从而改善机体本身的炎性环境。。
在本发明中，编码白细胞介素10的核酸和编码白细胞介素37的核酸包含于相同或不同的表达载体中，优选包含于相同的表达载体中。
在进一步优选的实施方式中，所述编码白细胞介素10的核酸和编码白细胞介素37的核酸通过编码自我切割肽的序列连接，例如具有如SEQ ID NO：3所示序列的核酸，其编码具有如SEQ ID NO：3所示氨基酸序列的T2A多肽。
所述编码自我切割肽的序列连接在所述编码白细胞介素10的核酸的3'端和编码白细胞介素37的核酸的5'端之间。
在本发明一个优选实施方式中，所述编码抗炎因子的核酸通过易在干细胞中沉默的强启动子携带，主要目的是为了使其在干细胞移植初期大量分泌炎性抑制因子从而起到改善机体微环境中炎症特征，而利用其易沉默的特点使其在后续过程中逐渐沉默，从而避免过度的免疫抑制。
作为强启动子，优选采用CMV，其能高效快速驱动诱导抗炎因子表达，迅速形成炎性抑制环境，提供良好的治疗环境。
所述强启动子连接在编码白细胞介素37的核酸的5'端。
在根据本发明提供的基因修饰干细胞的另一个优选实施方式中，所述软骨修复因子选自成纤维细胞生长因子如FGF2a、FGF2b、FGF18或FGF23等，更优选地，所述软骨修复因子选自成纤维细胞生长因子18(FGF18)。
优选地，编码成纤维细胞生长因子18的核酸具有如SEQ ID NO：4所示的氨基酸序列。
成纤维细胞生长因子(FGFs)是由约150-200氨基酸组成的多肽，以两种密切相关的形式存在，即碱性成纤维细胞生长因子(bFGF)与酸性成纤维细胞生长因子(aFGF)。两种FGF 都刺激分离大鼠颅盖骨成骨细胞样细胞的DNA合成，同时bFGF刺激分化软骨细胞在琼脂中的集落形成，起丝裂原和形态作用。FGF在活体内增加分化分化较差的前成骨细胞的数量。将FGF加入鸡胚，可使成骨细胞、软骨细胞增殖及骨基质形成。
FGF18是一个由207氨基酸组成的高度保守蛋白，与已发现的FGFs其他成员有30％-70％的同源性。FGF18蛋白在骨骼生长发育和发展过程中扮演着重要的角色，同时也参与到皮质神经元活动，腺垂体的生存、分化和增殖，以及调节头发的生长和皮肤修复。
本发明人发现，在骨膝关节治疗的时候，治疗部位往往存在严重的炎症反应，特别是用干细胞进行损伤修复治疗后，部分患者更会出现损伤部位僵直疼痛加剧的症状。在临床上多采用封闭疼痛性药物来改善该症状。另外，干细胞的定向分化是根据微环境而不同的，炎性环境的存在并不利于干细胞向骨和软骨方向分化。本发明人发明，通过让干细胞自身持续分泌抗炎因子如IL10和IL37，改善自身分化微环境，持续缓解局部注射炎症反应，然后结合驱动干细胞向软骨分化的因子(FGF18)，使得干细胞能够更好的向骨和软骨方向分化，这样就降低了体外持续给药反复刺激局部部位的影响。在本发明一个优选实施方式中，所述编码软骨修复因子的核酸采用不易在干细胞中沉默的中等强度启动子携带，其有助于在初期迅速形成的炎性抑制环境下稳定地表达软骨修复因子，获得稳定的软骨修复效果。
作为中等强度启动子，优选采用EF1α、SV40、PGK1或Ubc等，优选EF1α启动子，因其是来源于人延长因子1α的强哺乳动物细胞表达启动子，其表达水平十分稳定，与细胞类型和细胞所处使其无关。
所述中等强度启动子优选通过增强子连接在编码软骨修复因子的核酸的5'端。
根据本发明一种优选的实施方式，所述基因修饰的干细胞还包括表达载体，编码抗炎因子的核酸和编码软骨修复因子的核酸包含于相同或不同的表达载体中，优选包含于相同的表达载体中。
在进一步优选的实施方式中，所述编码抗炎因子的核酸和编码软骨修复因子的核酸通过中等强度启动子连接。
在本发明一个优选实施方式中，编码抗炎因子的核酸和编码软骨修复因子的核酸连接，例如通过中等强度启动子连接。
更优选地，中等强度启动子如EF1a连接增强子，增强子再连接编码软骨修复因子的核酸的5'端，中等强度启动子再连接编码抗炎因子的核酸。
根据本发明一种优选的实施方式，所述干细胞为间充质干细胞，来源于脂肪组织、脐带、骨髓或脐血，优选来源于脐带。
其中，所述间充质干细胞(MSC)是一种多能干细胞，它具有干细胞的所有共性，即自我更新和多向分化能力。此外，其具有免疫调节功能，通过细胞间的相互作用及产生细胞因子抑制T细胞的增殖及其免疫反应，从而发挥免疫重建的功能；同时，间充质干细胞来源方便，易于分离、培养、扩增和纯化，多次传代扩增后仍具有干细胞特性，不存在免疫排斥的特性。
脐带间充质干细胞来源于离体的脐带组织，取材方便，来源广泛，不受伦理的争论和限制，同时取材对供者无创伤，不受供者年龄因素的影响，体外分离培养简单，扩增迅速，免疫源性较低，无致瘤性，可作为基因治疗的细胞子载体。在体内或体外特定的诱导条件下，脐带间充质干细胞可分化为多种组织细胞，连续传代培养和冷冻保存后仍具有多向分化潜能，是细胞移植治疗的理想种子细胞。
因此，本发明中优选采用脐带来源的间充质干细胞。
二、关于基因修复的间充质干细胞制备方法
本发明提供用于软骨修复治疗的基因修饰干细胞的制备方法，包括将编码抗炎因子的核酸和编码软骨修复因子的核酸连接，并导入间充质干细胞。
根据本发明的一种优选的实施方式，所述制备方法包括以下步骤：
步骤1，获得编码抗炎因子的核酸和编码软骨修复因子的核酸，酶切后与载体质粒连接，得到重组质粒；
步骤2，将重组质粒与包装质粒进行慢病毒包装细胞的转染，得到含有编码抗炎因子的核酸和编码软骨修复因子的核酸的重组慢病毒载体；
步骤3，将上述获得的重组慢病毒载体转染至间充质干细胞，得到用于软骨修复治疗的基因修饰的干细胞。
以下进一步描述基因修饰的干细胞的制备方法：
步骤1，获得编码抗炎因子的核酸和编码软骨修复因子的核酸，酶切后与载体质粒连接，得到重组质粒。
其中，所述编码抗炎因子的核酸为编码双抗炎因子的核酸，能同时编码IL10和IL37的核酸，其编码IL10的部分具有如SEQ ID NO：1所示的核苷酸序列；其编码IL37的部分具有如SEQ ID NO：2所示的核苷酸序列。
在该编码双抗炎因子的核酸中，编码IL10的核酸和编码IL37的核酸优选通过T2A序列连接，所述T2A序列如SEQ ID NO:3所示。
该编码双抗炎因子的核酸中编码IL10的部分与强启动子CMV连接。
所述编码软骨修复因子的核酸为编码FGF18的核酸，具有如SEQ ID NO：4所示的核苷酸序列。
在优选的实施方式中，编码FGF18的核酸5’端通过增强子5'LTR序列连接中等强度启动子，例如具有如SEQ ID NO：7所示核苷酸序列的EF1a序列。
在更优选的实施方式中，该编码抗炎因子的核酸中编码IL37的部分通过启动子EF-1α与增强子5'LTR序列(其具有如SEQ ID NO：5所示核苷酸序列)连接，5'LTR序列再与编码软骨修复因子的核酸连接。
在本发明中，采用现有技术中常用的方法包括编码抗炎分子的核酸部分与编码软骨修复因子的核酸部分的码核酸进行酶切，并对载体质粒进行酶切，用T4连接酶进行4℃过夜连接，转化DH5α感受态细胞，取100μL菌液涂布至含有氨苄霉素抗性的LB板上，37℃过夜培养，挑取单克隆进行菌落PCR，将阳性克隆送样测序，保存测序结果正确的克隆并提取质粒，其图谱如图1所示。
其中，所述载体质粒为pCDH-CMV，重组质粒为pCDH-103718，酶切采用的酶优选为XbaI和SaII。
步骤2，将重组质粒与包装质粒进行慢病毒包装细胞的转染，得到含有编码抗炎分子的核酸和编码软骨修复分子的核酸的重组慢病毒载体。
其中，步骤2包括以下子步骤：
步骤2-1，培养包装细胞
在本发明中，所述包装细胞优选为293T细胞，将293T细胞活化后重悬，接种至培养皿中培养，待细胞汇合度达90％以上时，进行消化；终止消化后，对细胞进行离心、重悬，在每个包被过的培养皿中接种细胞用于包装病毒，优选每个培养皿(150mm)接种1×10 7～1.5×10 7个细胞，优选每个培养皿接种1.2×10 7个细胞。
其中，293T细胞的培养基为10％FBS+1mM丙酮酸钠+2mM谷氨酰胺+1％非必需氨基酸+DMEM培养基。
步骤2-2，转染包装细胞
具体地，将包装质粒与步骤1获得的重组克隆质粒(pCDH-103718)混合，温育后加入转染试剂，室温孵育，形成DNA-转染试剂复合物。
在本发明中，所述包装质粒可以为现有技术中常用的慢病毒包装辅助质粒，如购自Addgene的PMD.2G和PSPAX，优选PMD.2G质粒和PSPAX质粒的质量比为2:1，更优选 PMD.2G质粒、PSPAX质粒和pCDH-103718质粒的质量比为2:1:3。
所述转染试剂可以为现有技术中常用的试剂，例如可以为PEI(购自Polyscience)溶液，转染试剂与DMEM培养基混合后成滴的加入到上述质粒混合体系中。
将DNA-转染试剂复合物与包装细胞混合均匀，培养，培养过程中定时换液，换液后收集上清液体存储于4℃。
根据本发明一种优选的实施方式，对收集的上清液体进行浓缩，并进行病毒滴度测定，
所述浓缩优选为对上清液体低温离心。
步骤3，将上述获得的重组慢病毒载体转染至间充质干细胞，得到基因修饰的干细胞。
根据本发明一种优选的实施方式，所述干细胞为间充质干细胞，来源于脂肪组织、脐带、骨髓或脐血，优选来源于脐带。
优选地，采用脐带组织块爬片法分离间充质干细胞。
具体地：将正常分娩的离体脐带放入含有200U/mL青霉素和200U/mL链霉素的PBS缓冲液中，为保证脐带组织活性，新鲜脐带需在6h内分离完毕。用20mL注射器冲洗脐静脉和脐动脉内的残存积血，用组织剪将脐带组织剪碎成1mm 3大小的组织块，再将获得的小块脐带组织用200目滤网滤过，收集200目滤网上的脐带组织块，去掉过小的脐带组织块，获得直径为1-1.5mm多个脐带组织块。收集直径为1-1.5mm的组织块，将组织块直接接种在培养瓶中，直接放置于5％CO 2、37℃培养箱内，静置1-2h。待组织块贴壁比较牢固后，添加含10％胎牛血清的α-MEM培养液，置于5％CO 2、37℃培养箱内继续培养，五天后，在培养瓶中脐带组织间充质干细胞增生铺满约80％；以0.25％胰蛋白酶(0.01％EDTA)消化，所得细胞为原代细胞。脐带组织块爬片法分离培养MSC，72h后在脐带组织周围有少量细胞爬出，约7天后，细胞游离出组织，并逐渐形成克隆，制得的间充质干细胞冻存。
步骤3包括以下子步骤：
步骤3-1，对干细胞进行培养，接种
对预先冻存的干细胞进行复苏培养，待复苏细胞长满后，0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，终止消化后，离心、重悬细胞。然后接种细胞至培养皿，优选每个培养皿(150mm)接种2×10 6～2.5×10 6个细胞，接种后第二天更换培养基。
步骤3-2，加入慢病毒载体，继续培养
向接种后的细胞中加入聚凝胺(Polybrene)，根据细胞MOI值及病毒滴度，加入相应体积的慢病毒载体LV-103718慢病毒，进行培养。
其中，加入的慢病毒载体体积＝(MOI×细胞数目)/病毒滴度。所述MOI指的是转导时病毒与细胞数量的比值，在本发明中，所述MOI优选为10～50，如40。
在37℃、5％CO 2饱和湿度培养6-8h，然后弃掉含有病毒的培养基，更换为无血清培养基，37℃、5％CO2饱和湿度继续培养2-3天。
步骤3-3，对细胞进行传代培养
在本发明中，待基因修饰后的细胞长满，采用0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，细胞悬液800rpm离心5min，离心所得细胞用无血清培养基重悬，按照1:6的传代比例进行传代，无血清培养基37℃、5％CO 2培养3天至细胞长满，获得基因修饰的干细胞，记为MSC-103718。
再者，本发明还提供了上述方法制备得到的基因修饰的干细胞。
又者，本发明还提供了上述基因修饰的干细胞在制备用于软骨疾病修复/治疗的药物中的用途。
其中软骨性疾病包括急性软骨损伤、慢性关节软骨磨损、复发性多软骨炎等，优选为慢性关节软骨磨损。
典型地，软骨性疾病表现为退行性关节炎、滑囊炎、滑膜炎、颈椎病、腰椎病、肩周炎、骨质增生、风湿性关节炎和类风湿性关节炎等。
又再者，本发明还提供了一种药物组合物，其包括上述基因修饰的干细胞或通过上述方法制备的干细胞。
其中，所述药物组合物的剂型可以为医学领域已知的任何形式，优选为片剂、丸剂、混悬剂、乳剂、溶液、注射剂、凝胶剂、胶囊剂、粉剂、颗粒剂或栓剂，更优选为注射剂。
优选地，所述药物组合物还包括药学上接受的载体或赋形剂，优选包括药学上可接受的无菌等渗水性或非水性溶液(例如：平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
更优选地，所述药物组合物可以以悬浮液、凝胶、胶体、浆液或混合物的形式进行移植。
在本发明中，所述药物组合物的配置按照现有技术中规定的细胞药物的原则进行，例如可以按照《造血干细胞疗法(HematopoieticStemCellTherapy)》，E.D.Ball，J.Lister&P.Law，ChurchillLivingstone，2000进行。
根据本发明一种优选的实施方式，所述药物组合物可以通过皮内注射、皮下注射、肌肉注射、静脉注射或口服的方式施用给受试者，
所述受试者是哺乳动物，例如人。
实施例
以下通过具体实例进一步描述本发明，不过这些实例仅仅是范例性的，并不对本发明的保护范围构成任何限制。
实施例1
合成人源XbaI-IL10-T2A-IL37-EF1α-5'LTR-FGF18-SaIl
将IL10编码核苷酸(核苷酸序列为SEQ ID NO:1所示)和IL37编码核苷酸(核苷酸序列为SEQ ID NO:2所示)用T2A(苷酸序列为SEQ ID NO:3所示)连接，FGF18编码核苷酸(苷酸序列为SEQ ID NO:4所示)前连接5'LTR序列(序列如SEQ ID NO：5所示)和EF1α启动子(序列如SEQ ID NO：7所示)，IL37与EF1α启动子连接，两端引入酶切位点XbaI和SaII，合成人源XbaI-IL10-T2A-IL37-EF1α-5'LTR-FGF18-SaIl，命名为Sequence103718，其序列如SEQ ID NO：6所示。
实施例2 构建pCDH-103718质粒
将pCDH-CMV质粒(Addgene)用XbaI和SaII酶切，产物胶回收6195bp左右片段作为载体。
将Sequence103718用XbaI和SaII酶切，产物胶回收2487bp左右片段。
取pCDH-CMV质粒酶切产物载体与Sequence103718酶切产物片段，用T4连接酶进行4℃过夜连接，转化DH5α感受态细胞，取100μL菌液涂布至含有氨苄霉素抗性的LB板上，37℃过夜培养，挑取单克隆进行菌落PCR，将阳性克隆送样测序，保存测序结果正确的克隆并提取质粒，命名为pCDH-103718，其图谱如图1所示。
实施例3 pCDH-103718质粒慢病毒包装
(1)293T细胞准备：从液氮中取出1支冻存的293T细胞(购自ATCC)迅速放到37℃水浴中直至冰块消失，逐滴加入含有5ml预热培养基的15ml离心管中，1200rpm离心3min，弃上清，用293T培养基(10％FBS+1mM丙酮酸钠+2mM谷氨酰胺+1％非必需氨基酸+DMEM)重悬细胞接种至150mm培养皿中，37℃、5％CO 2饱和湿度培养。培养过程中，待细胞汇合度达90％以上时，进行传代培养，弃去旧培养基，加入5ml灭菌PBS溶液，轻轻晃动，洗涤细胞后弃去PBS溶液，加入2ml0.25％Trypsin-EDTA消化液，消化1-2min直到细胞完全消化下来。加入含血清的培养基终止消化，细胞悬液1200rpm离心3min，离心所得细胞用培养基重悬。每个包被过的150mm培养皿细胞接种1.2×107细胞用于包装慢病毒，37℃、5％CO 2饱和湿度培养，20ml培养基/皿。
(2)转染293T细胞：转染前2小时，将293T细胞培养基更换为18mlDMEM培养基，向A灭菌离心管中加入1ml预热的DMEM培养基，按照质量比例2:1:3加入包装质粒PMD.2G (Addgene)和PSPAX(Addgene)和质粒pCDH-103718的混合物，吹打混匀。向B灭菌离心管中加入1ml预热的DMEM培养基，然后加入162μl转染试剂PEI，混合均匀。A管和B管在室温下温育5min。将B管中的液体成滴地加入到A管中，混合均匀，室温孵育10min，以便形成DNA-转染试剂复合物。将DNA-转染试剂复合物转移至预先换液的293T细胞中，混匀，37℃、5％CO 2饱和湿度培养。培养6-8h后吸弃含有转染混和物的培养基，每皿细胞加入20ml预热的含5％FBS的DMEM培养基，37℃、5％CO 2饱和湿度培养。换液后分别在24h和48h收集上清液暂存储于4℃，并换20ml新鲜培养基。
(3)重组慢病毒收集与浓缩：将收集到的液体4℃、3500rpm离心15min，弃沉淀，将上清与5XPEG混匀，4℃放置24小时后，4℃3000rpm离心30min，弃上清，将沉淀重悬至500μlDMEM培养基中，进行病毒滴度测定，病毒命名为LV-103718。
实施例4 间充质干细胞的分离培养
采用脐带组织块爬片法分离间充质干细胞，具体步骤如下：
将正常分娩的离体脐带放入含有200U/mL青霉素和200U/mL链霉素的PBS缓冲液中，为保证脐带组织活性，新鲜脐带需在6h内分离完毕。用20mL注射器冲洗脐静脉和脐动脉内的残存积血，用组织剪将脐带组织剪碎成1mm3大小的组织块，再将获得的小块脐带组织用200目滤网滤过，收集200目滤网上的脐带组织块，去掉过小的脐带组织块，获得直径为1-1.5mm多个脐带组织块。收集直径为1-1.5mm的组织块，将组织块直接接种在培养瓶中，直接放置于5％CO 2、37℃培养箱内，静置1-2h。待组织块贴壁比较牢固后，添加含10％胎牛血清的α-MEM培养液(购自Gibco)，置于5％CO 2、37℃培养箱内继续培养，五天后，在培养瓶中脐带组织间充质干细胞增生铺满约80％；以0.25％胰蛋白酶(0.01％EDTA)消化，所得细胞为原代细胞。脐带组织块爬片法分离培养MSC，72h后在脐带组织周围有少量细胞爬出，约7天后，细胞游离出组织，并逐渐形成克隆，制得的间充质干细胞冻存。
实施例5 间充质干细胞的基因修饰
复苏预先冻存的P3代间充质干细胞至一个150mm培养皿，20ml无血清培养基37℃、5％CO 2饱和湿度培养。待复苏细胞长满后，0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，细胞悬液800rpm离心5min，离心所得细胞用MSC无血清培养基(购自Bioind)重悬。每个150mm培养皿细胞接种2-2.5×10 6细胞，接种后的第二天吸取细胞的培养基弃掉，更换为无血清的α-MEM培养基，20ml培养基/皿，加入16μlPolybrene(购自Sigma)，按照40MOIs感染复数同时加入实施例4中获得的LV-103718慢病毒(滴度为1×10 8U/ml)，37℃、5％CO 2饱和湿度培养6-8h。6-8小时后弃掉含有病毒的α-MEM培养基，更换为无血清培养基，37℃、5％CO 2饱和湿度继续培养2-3天。待细胞长满，0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，细胞悬液800rpm离心5min，离心所得细胞用无血清培养基重悬，按照1:6的传代比例进行传代，无血清培养基37℃、5％CO 2培养3天。所得LV-103718修饰的间充质干细胞命名为MSC-103718。
实验例
实验例1 细胞表型的鉴定
选择冻存前细胞，用0.05％胰酶消化，PBS洗两遍后分别用小鼠抗人CD11b-PE、CD45-PE、HLA-DR-PE、CD73-PE、CD90-PE、CD105-PE、CD34-FITC及CD19-FITC抗体标记5×10 5个MSCs，室温避光放置30min，再用PBS洗两遍后，4％多聚甲醛固定，FACS检测。鉴定合格的细胞冻存于液氮罐中，用时复苏并做后期处理。具体如图2所示。
结果显示：基因修饰并不影响MSC细胞的干性，对后期向骨和软骨分化无明显不良影响。
实验例2 MSC-103718细胞中IL10、IL37和FGF18的表达量的检测
在100mm培养中，分别培养不同组别细胞，当细胞汇合度达70％-80％时，吸弃原有MSC无血清培养基，10mlα-MEM培养基，37℃、5％CO 2饱和湿度继续培养48h。收集三种 细胞的培养上清4℃保存备用。长期保存需放置于-80℃冰箱。用人源IL10、IL37和FGF18检测试剂盒(欣博盛生物科技有限公司)按照说明书检测MSC-103718细胞中IL10、IL37和FGF18因子的分泌量。
结果如图3所示，LV-103718基因修饰后的MSC在可高表达IL10、IL37和FGF18因子。
实验例3 成脂分化、成骨分化和成软骨分化
(1)成脂诱导
以维持培养基接种hMSC：用MSCAttachmentsolution(BI；P/N:05-752-1，1:100DPBS稀释)预包被24孔盘，每孔加入0.5mlMSCNutriStem
Figure PCTCN2022109763-appb-000001
XF，接种6x104细胞(3x104cells/cm2)。置于37℃，5％CO 2细胞培养箱中培养。
以成脂分化培养基诱导分化：培养24h后确认细胞融合度达到80-90％，将维持培养基吸除，每孔(24孔盘)加入0.5ml分化培养基。置于37℃，5％CO 2细胞培养箱中培养14-21天。期间换液操作如下，使用完全分化培养基培养6-8天，期间每3-4天换液。分化完成后，换液成维持培养基。观察到成熟脂肪细胞(即脂滴的形成)，即可染色。
Oilred-O染色过程：吸除培养基，用DPBS洗一次(1ml/well,24孔盘)。固定：吸除DPBS，加入10％福尔马林(4％甲醛；1ml/well，24孔盘)。室温固定30-60分钟。吸除福尔马林，用60％的异丙醇洗涤2-3分钟(1ml/well，24孔盘)。吸除异丙醇，加入Oilred-O染色工作液(1ml/well，24孔盘)。室温静置10-30分钟。用蒸馏水洗涤，去除多余的染料。染色效果如图4所示，其中，样本1和样本2是来源不同个体的两个独立样本。
(2)成骨诱导
接种hMSC：用MSCAttachmentsolution(BI；P/N:05-752-1，1:100DPBS稀释)预包被24孔盘，每孔加入0.5mlMSCNutriStem
Figure PCTCN2022109763-appb-000002
XF，接种6x10 4细胞(3x10 4cells/cm 2)。置于37℃，5％CO 2细胞培养箱中培养。
以成骨分化培养基诱导分化：培养24h后确认细胞融合度达到80-90％，将维持培养基吸除，每孔(24孔盘)加入0.5ml分化培养基。置于37℃，5％CO 2培养箱中培养10-21天，每2-3天换液。
成骨评价：吸除培养基，用DPBS(BI；02-023-1)洗一次(1ml/well)。固定：吸除DPBS，每孔加入1ml70％EtOH。室温固定30-60分钟。吸除EtOH，用蒸馏水洗涤3次(1ml/well)。吸除蒸馏水，每孔加入1ml2％ARS染色工作液。室温静置30-60分钟。吸除染色液，用1ml蒸馏水洗涤4次(1ml/well)。每孔加入1ml蒸馏水避免细胞干燥。成骨染色效果如图5所示，其中，样本1和样本2是来源不同个体的两个独立样本。
(3)成软骨诱导
以维持培养基接种hMSC：细胞以1x10 5/(10μl培养基)接种进1孔U底的96孔盘(非TC-treated)置于37℃，5％CO 2细胞培养箱2小时后(促进细胞聚集成团)，小心加入0.1ml培养基。小微团的培养方式有助于球状细胞团形成，放回37℃，5％CO 2细胞培养箱继续培养。
以成软骨分化培养基诱导分化：培养24小时后，将维持培养基吸除，每孔(96孔盘)加入0.2ml分化培养基，球状细胞团会在24-48小时形成。置于37℃，5％CO 2细胞培养箱中培养14-21天。间每3-4天更换一次分化培养基。培养时间长有助于获得更多成熟的软骨细胞。
AlcianBlue染色过程：吸除培养基，用DPBS洗一次(BI；02-023-1，0.2ml/well)。固定：吸除DPBS，每孔加入0.2ml的10％福尔马林(4％甲醛)。室温固定30-60分钟。吸除福尔马林，每孔用0.2ml蒸馏水洗涤2次。吸除蒸馏水，每孔加入0.2ml的AlcianBlue染色工作液。室温避光过夜。吸除染液，每孔用0.2ml0.1NHCl洗涤2-3次。吸除HCl，每孔加入0.2ml蒸馏水。染色结果如图6和图7所示，其中，样本1-样本4是来源不同个体的四个独立样本。
由如图4、图5、图6和图7可知，LV-103718修饰后的干细胞MSC-103718可迅速分化为 成骨细胞和成软骨细胞，且效率要明显高于未修饰的MSC细胞；LV-103718修饰对成脂分化无显著影响。
实验例4 MSC-103718对于炎性软骨恢复效果实验
(1)免疫学反应检测
PBMC分离培养：将10ml全血转入50ml离心管中，加入10mlPBS溶液稀释，轻轻混匀；取两支15ml离心管，先加入5ml淋巴细胞分离(ficoll)液。然后将稀释的血液轻轻加到两支离心管的ficoll上层，一定要轻柔，避免两种溶液混合在一起，每只离心管各10ml稀释血液；2,000rpm，20min，注意，降速设置中一定要设置成nobreak，或者只有1-2成的制动。离心完毕；PBMC所在细胞层为白色。此时可以用吸管将该层细胞吸取在另一干净的15ml离心管中。加入PBS至10-15ml，1,500rpm，10min离心后去掉上清，再加入培养基进行相同操作的清洗；加入5-10ml培养基重悬细胞，进行后续计数培养或者铺板。
MSCs(MSC组，MSC-1037组，和MSC-18组)与PBMC共培养：各将生长状态良好的各组MSC(MSC组、MSC-1037(以实施例1-4的方法获得，区别在于实施例1中合成人源 XbaI-IL10-T2A-IL37-EF1α-SaIl)组、MSC-18(以实施例1-4的方法获得，区别在于实施例1中合成人源 XbaI-EF1α-5'LTR-FGF18-SaIl)组和MSC-103718组)计数，离心重悬调整细胞浓度到3×10 5/ml，各按2ml/孔接入6孔板中，平行3个孔，37℃，5％CO2培养16-18h细胞稳定贴壁后换液，按MSCs：PBMC＝1:5的比例进行混合，置于37℃、5％CO 2培养箱中继续共培养。48h后，收集细胞悬液用流式细胞术检测相应免疫细胞表型，收集培养上清用ELISA检测TNF-α细胞因子的含量。
结果，如图8所示：相比其它各组，MSC-103718组能够更好的抑制总淋巴细胞增殖、抑制Th1型和Th17型淋巴细胞增殖，促进Treg细胞增殖；如图9所示：相比其它各组，MSC-103718组能够更好的抑制TNF-α细胞因子的表达。
(2)软骨修复能力向软骨定向分化培养：P1传代细胞培养至细胞铺满瓶底时，消化后调整细胞浓度，加入含TGFβ1(10ng/ml)、10％胎牛血清的高糖DMEM溶液诱导培养。将不同组的MSC组、MSC-1037组、MSC-18组和MSC-103718组的细胞悬液调整浓度后，滴加到PLGA支架上，37℃、5％CO 2孵箱培养7～d。复合体移植10～12个月龄健康比格犬10只，静脉麻醉成功后，术区准备完毕后，取膝关节内侧切口，显露膝关节，在膝关节股骨滑车处，用手摇钻制作一个直径为mm的圆柱形软骨缺损区，深达软骨下骨。大体观察分别于术后12、16周取材，观察缺损区软骨生长情况。
结果：复合材料显示MSCs细胞在PLGA支架上的黏附、伸展和增殖良好，可以见到细胞分泌的基质和细胞伪足的铆固作用，表明支架材料具有良好的细胞亲和性。
术后12周：MSC-103718组新生组织表面平整、光滑，与周围软骨界线模糊，端面显示软骨厚度与正常软骨相近；MSC和MSC-1037组修复高度较周围软骨水平相近，但软骨层厚度比A组明显偏薄，无光泽，与周围软骨界线尚清楚；MSC-18组部分接近正常修复高度，修复组织呈黄色，局部凹陷。术后16周，MSC-103718组缺损修复区组织与周围关节软骨相整合，软骨缺损区被光滑白色半透明的组织覆盖，与周围软骨组织外形无差异；MSC-Con和MSC-1037组缺损区修复组织与周围软骨部分整合在一起，光泽较差；MSC-18组缺损处修复组织低凹新生组织软，无光泽，与周围软骨组织区别明显。
SEQ ID NO:1编码IL10的核苷酸序列(534bp)
Figure PCTCN2022109763-appb-000003
Figure PCTCN2022109763-appb-000004
SEQ ID NO:2编码IL37的核苷酸序列(654bp)
Figure PCTCN2022109763-appb-000005
SEQ ID NO:3 T2A核苷酸序列(54bp)
Figure PCTCN2022109763-appb-000006
SEQ ID NO:4编码FGF18的核苷酸序列(621bp)
Figure PCTCN2022109763-appb-000007
SEQ ID NO:5 5’LTR核苷酸序列(269bp)
Figure PCTCN2022109763-appb-000008
SEQ ID NO:6 XbaI-IL10-T2A-IL37-EF-1α-5'LTR-FGF18-SaIl的核苷酸序列(2506bp)
Figure PCTCN2022109763-appb-000009
Figure PCTCN2022109763-appb-000010
SEQ ID NO:7EF1a核苷酸序列(212bp)
Figure PCTCN2022109763-appb-000011
以上结合具体实施方式和范例性实例对本发明进行了详细说明，不过这些说明并不能理解为对本发明的限制。本领域技术人员理解，在不偏离本发明精神和范围的情况下，可以对本发明技术方案及其实施方式进行多种等价替换、修饰或改进，这些均落入本发明的范围内。

Claims (10)

  1. 一种用于软骨修复治疗的基因修饰干细胞，所述干细胞包括编码抗炎因子的核酸和编码软骨修复因子的核酸。
  2. 如权利要求1所述用于软骨修复治疗的基因修饰干细胞，其中所述抗炎因子选自白细胞介素和TGFβ受体家族，优选地，所述抗炎因子选自白细胞介素-3(IL-3)、白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-13(IL-13)和白细胞介素-37(IL-37))中的两种或更多种，优选为白细胞介素10和白细胞介素37。
  3. 如权利要求1所述用于软骨修复治疗的基因修饰干细胞，其中所述软骨修复因子选自成纤维细胞生长因子，如FGF2a、FGF2b、FGF18或FGF23等，优选地，所述软骨修复因子选自成纤维细胞生长因子18(FGF18)。
  4. 如权利要求1所述用于软骨修复治疗的基因修饰干细胞，所述编码抗炎因子的核酸通过易在干细胞中沉默的强启动子携带，所述编码软骨修复因子的核酸采用不易在干细胞中沉默的中等强度启动子携带。
  5. 如权利要求1所述用于软骨修复治疗的基因修饰干细胞，编码抗炎因子的核酸和编码软骨修复因子的核酸连接。
  6. 用于软骨修复治疗的基因修饰干细胞的制备方法，包括将编码抗炎因子的核酸和编码软骨修复因子的核酸连接，并导入间充质干细胞。
  7. 如权利要求6所述用于软骨修复治疗的基因修饰干细胞的制备方法，包括以下步骤：
    步骤1，获得编码抗炎因子的核酸和编码软骨修复因子的核酸，酶切后与载体质粒连接，得到重组质粒；
    步骤2，将重组质粒与包装质粒进行慢病毒包装细胞的转染，得到含有编码抗炎因子的核酸和编码软骨修复因子的核酸的重组慢病毒载体；
    步骤3，将上述获得的重组慢病毒载体转染至间充质干细胞，得到用于软骨修复治疗的基因修饰的干细胞。
  8. 通过权利要求6或7所述方法制备得到的基因修饰的干细胞。
  9. 权利要求1-5任一项所述基因修饰干细胞或通过权利要求6或7所述方法制备得到的基因修饰的干细胞在制备用于软骨性疾病的修复/治疗药物中的用途。
  10. 如权利要求9所述的用途，其中软骨性疾病包括退行性关节炎、滑囊炎、滑膜炎、颈椎病、腰椎病、肩周炎、骨质增生、风湿性关节炎和类风湿性关节炎等。
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