Preparation method and application of genetically modified mesenchymal stem cells

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a genetically modified mesenchymal stem cell as well as a preparation method and application thereof. The mesenchymal stem cells are modified by adopting the GDF5 gene and the anti-MMP9 nano antibody, so that not only is the proliferation activity of the mesenchymal stem cells improved, but also the inflammatory response of arthritis is reduced, the pain caused by the arthritis is relieved, and the arthritis is effectively treated.

Description

Technical Field

[0001] The present invention relates to mesenchymal stem cells, and in particular to a preparation method and application of gene-modified mesenchymal stem cells. Background Art

[0002] In regenerative medicine and clinical treatment, mesenchymal stem cells (MSCs) have become a promising alternative to embryonic stem cells due to their ability to self-renew and multi-lineage differentiation. In recent years, clinical studies have shown that mesenchymal stem cells have significant therapeutic effects on immune diseases, myocardial injury, liver disease, lung disease, kidney disease and diabetes. MSCs are an important cell bank involved in tissue regeneration. Under the action of special signals caused by tissue damage, MSCs migrate to the damaged area, aggregate and proliferate locally, and differentiate along different pathways according to different damage signals.

[0003] MSCs are easy to separate and proliferate, but their proliferation ability is affected by many factors, including the individual tissue they are derived from, culture conditions, and continuous passage. The rate of mesenchymal stem cells proliferating in vitro without genetic modification is slow, and it takes a long time to expand to the level of clinical application. Therefore, finding and cloning genes related to the expression and regulation of the proliferation ability of mesenchymal stem cells is not only beneficial to the proliferation of mesenchymal stem cells, but also conducive to the wide application of clinical treatment.

[0004] Growth / Differentiationfactor5 (GDF5) is a member of the bone morphogenetic protein family and transforming growth factor β superfamily, also known as chondrogenic protein (CDMP-1) or bone morphogenetic protein 14 (BMP-14). As one of the cytokines of tissue engineering, it can not only promote the proliferation of mesenchymal stem cells, but also promote the differentiation of mesenchymal stem cells, and has a good repair effect on cartilage and bone damage.

[0005] Arthritis is a degenerative disease of articular cartilage, accompanied by subchondral bone sclerosis, bone cystic changes and synovitis, which together promote the occurrence and development of arthritis. Studies have shown that GDF5 is a protective factor for the development of arthritis, and exogenous addition of GDF5 can reduce the progression of arthritis. In addition, studies have also shown that matrix metalloproteinase 9 (MMP9) is closely related to the occurrence and development of arthritis.

[0006] Currently, there are no studies on the combined use of GDF5 and MMP9 to treat arthritis. Therefore, research is needed to effectively treat arthritis. Summary of the invention

[0007] Based on the deficiencies in the prior art, the purpose of the present invention is to combine the two targets of GDF5 and MMP9, modify the GDF5 gene and anti-MMP9 nanoantibodies onto mesenchymal stem cells, and treat arthritis.

[0008] One aspect of the present invention is to provide a mesenchymal stem cell, specifically, the GDF5 gene and the anti-MMP9 nanobody are modified on the mesenchymal stem cell.

[0009] Preferably, the amino acid sequence of the GDF5 gene is shown as SEQ ID No.1, and the nucleotide sequence thereof is shown as SEQ ID No.3.

[0010] Preferably, the amino acid sequence of the anti-MMP9 nanobody is shown as SEQ ID No.2, and the nucleotide sequence thereof is shown as SEQ ID No.4.

[0011] Preferably, the GDF5 gene is optimized based on GENBANK No. ADD39552.1.

[0012] Preferably, the anti-MMP9 nanobody is prepared by a conventional nanobody preparation method.

[0013] Another aspect of the present invention is to provide a method for preparing mesenchymal stem cells, the specific steps comprising:

[0014] S1. Preparation of mesenchymal stem cells: Take placenta, umbilical cord or bone marrow tissue containing primary mesenchymal stem cells, cut small pieces, wash with phosphate buffer on a culture dish until the washing solution is transparent, and then cut into pieces; transfer the cut tissue to a 50ml centrifuge tube, add 25ml collagenase IV with a mass / volume concentration of 0.1%, and shake and digest in a 37°C constant temperature box for 30min; add 20ml phosphate buffer to the digested tissue, mix well and filter through a 100μm mesh, centrifuge the filtrate at 1200 rpm in a 4°C centrifuge for 5min, remove the supernatant and retain the cell pellet; add 5ml of DMEM culture medium containing 20% ​​fetal bovine serum to the cell pellet, mix well and inoculate into a cell bottle, culture at 37°C, 5% CO2, change the medium every 2 days, and perform passaging;

[0015] S2. Construct a lentiviral vector; directly synthesize the GDF5 and anti-MMP9 nanoantibody nucleotide sequences according to the nucleotide sequences of SEQ ID No.3 and SEQ ID No.4, connect them into the restriction endonucleic acid lentiviral expression vector GV367 (AgeI / NheI restriction endonucleic acid, element sequence Ubi-MCS-SV40-EGFP-IRES-puromycin), construct a recombinant plasmid, and transform DH5α competent cells for amplification. Extract the plasmid for sequencing verification, and save the successfully constructed plasmid; digest the 293T cells in the logarithmic phase to prepare a cell suspension and inoculate it on a 24-well culture plate, and culture it in a cell culture incubator until the cell confluence rate is about 70-80%, and co-transfect the 293T cells with plasmid DNA, packaging plasmid pHelper1.0 vector, envelope plasmid pHelper2.0 vector and LIPOFECTAMINE 3000. After culturing for 6 hours, replace the fresh culture medium, and continue to culture for 72 hours, collect the lentivirus-rich 293T cell supernatant, filter, centrifuge, and concentrate to obtain a high-titer virus concentrate;

[0016] S3, take MSCs within 5 generations in the logarithmic growth phase, inoculate them in 12 culture plates at 100,000 cells / well, 1 ml of culture medium per well. When the cell confluence is about 50%, remove the culture medium, wash with PBS and transfect the virus concentrate in step S2 for 10 hours. Then replace the culture medium and continue to culture, and pass it according to the conventional method. Use the culture medium containing puromycin for screening for 7 days, and change the medium every 2 days to obtain MSCs that stably express GDF5 and anti-MMP9 nanoantibodies.

[0017] Another aspect of the present invention is to provide an application of mesenchymal stem cells, specifically for the treatment of arthritis, by combining the two targets of GDF5 and MMP9, modifying GDF5 and anti-MMP9 nanoantibodies to mesenchymal stem cells, which can alleviate the symptoms of arthritis and thus treat arthritis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention modifies GDF5 and anti-MMP9 nanoantibodies to mesenchymal stem cells for the first time, wherein GDF5 can not only promote the proliferation of mesenchymal stem cells, but also serve as targets for treating arthritis together with MMP9, thereby improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Western blot of IL-10 and FOXP3 proteins DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0022] Example 1 Preparation of Mesenchymal Stem Cells Modified with GDF5 and Anti-MMP9 Nanobodies

[0023] S1. Preparation of primitive mesenchymal stem cells: Take bone marrow tissue containing primary mesenchymal stem cells, cut small pieces, wash with phosphate buffer on a culture dish until the washing solution is transparent, and then cut into pieces; transfer the cut tissue to a 50ml centrifuge tube, add 25ml collagenase IV with a mass / volume concentration of 0.1%, and shake and digest in a 37°C constant temperature incubator for 30min; add 20ml phosphate buffer to the digested tissue, mix well and filter through a 100μm mesh, centrifuge the filtrate at 1200 rpm in a 4°C centrifuge for 5min, remove the supernatant and retain the cell pellet; add 5ml of DMEM culture medium containing 20% ​​fetal bovine serum to the cell pellet, mix well and inoculate into a cell bottle, culture at 37°C, 5% CO2, change the medium every 2 days for passaging;

[0024] S2. Construct a lentiviral vector; directly synthesize the GDF5 and anti-MMP9 nanoantibody nucleotide sequences according to the nucleotide sequences of SEQ ID No.3 and SEQ ID No.4, connect them into the restriction endonucleic acid lentiviral expression vector GV367 (AgeI / NheI restriction endonucleic acid, element sequence Ubi-MCS-SV40-EGFP-IRES-puromycin), construct a recombinant plasmid, and transform DH5α competent cells for amplification. Extract the plasmid for sequencing verification, and save the successfully constructed plasmid; digest the 293T cells in the logarithmic phase to prepare a cell suspension and inoculate it on a 24-well culture plate, and culture it in a cell culture incubator until the cell confluence rate is about 70-80%, and co-transfect the 293T cells with plasmid DNA, packaging plasmid pHelper1.0 vector, envelope plasmid pHelper2.0 vector and LIPOFECTAMINE 3000. After culturing for 6 hours, replace the fresh culture medium, and continue to culture for 72 hours, collect the lentivirus-rich 293T cell supernatant, filter, centrifuge, and concentrate to obtain a high-titer virus concentrate;

[0025] S3. Preparation of genetically modified mesenchymal stem cells: Take MSCs within 5 generations in the logarithmic growth phase, inoculate them in 12 culture plates at 100,000 cells / well, and 1 ml of culture medium per well. When the cell confluence is about 50%, remove the culture medium, wash with PBS, and transfect the virus concentrate in step S2 for 10 hours. Then replace the culture medium and continue to culture, and pass it according to the conventional method. Use a culture medium containing puromycin for screening for 7 days, and change the medium every 2 days to obtain MSCs that stably express GDF5 and anti-MMP9 nanoantibodies.

[0026] Comparative Example 1 Preparation of GDF5-modified mesenchymal stem cells

[0027] The difference from Example 1 is that when constructing the lentiviral vector, only the GDF5 gene is introduced into the plasmid to construct the recombinant plasmid, and the other steps are the same.

[0028] Comparative Example 2 Preparation of Mesenchymal Stem Cells Modified with Anti-MMP9 Nanobodies

[0029] The difference from Example 1 is that when constructing the lentiviral vector, only the gene of the anti-MMP9 nanobody is introduced into the plasmid to construct the recombinant plasmid, and the other steps are the same.

[0030] Example 2: Treatment of arthritis with mesenchymal stem cells

[0031] 1. Construction of Arthritis Animal Model

[0032] 30 rats are divided into 5 groups, respectively PBS control group, unmodified mesenchymal stem cell treatment group, mesenchymal stem cell treatment group of comparative example 1, mesenchymal stem cell treatment group of comparative example 2 and mesenchymal stem cell treatment group of embodiment 1, 6 rats in each group. Each rat is intraperitoneally injected with 6% sodium pentobarbital (body mass × 0.01mL / kg) for anesthesia, and the hair at the left knee joint of the rat is shaved, and 75% alcohol is deiodinated 2 times after medical iodine tincture disinfection, and the knee joint is flexed 45 °, and the articular cavity is punctured from the outside of the inferior patellar ligament, and the needle is inserted and retracted 2mm after reaching the femoral condyle, and 8% sodium iodoacetate 2mg (dissolved in normal saline 50μL) is injected. Keep room temperature 25~27℃, allow rats to move freely and take in sufficient water and food, and inject penicillin intramuscularly for 3d to prevent infection.

[0033] 2. Drug Treatment and Total Protein Collection

[0034] After 7 days of modeling, the therapeutic drugs were injected into the joint cavity on the first day after surgery, wherein the PBS control group was injected with 150 μL PBS, the unmodified mesenchymal stem cell treatment group was injected with 150 μL unmodified mesenchymal stem cells, the mesenchymal stem cell treatment group of comparative example 1 was injected with 150 μL mesenchymal stem cells of comparative example 1, the mesenchymal stem cell treatment group of comparative example 2 was injected with 150 μL mesenchymal stem cells of comparative example 2, and the mesenchymal stem cell treatment group of example 1 was injected with 150 μL mesenchymal stem cells of example 1. The second to fourth rounds of therapeutic drug injections were performed on the 7th, 14th and 21st days after surgery, respectively, and the injection scheme was the same as the first round. The rats were killed by cervical dislocation 7 days after the fourth round of treatment, and the tissues around the left knee joint were removed to extract total protein.

[0035] 3. Effect evaluation

[0036] Western blot was used to qualitatively and quantitatively analyze IL-10 and FOXP3 proteins to evaluate the effect of mesenchymal stem cells in the treatment of arthritis.

[0037] Example 3 Results Analysis and Conclusion

[0038] 3.1 Mesenchymal stem cells were transfected with lentiviral vectors. Under fluorescence microscopy, the mesenchymal stem cells were all green. This is because the lentiviral vector contains the GFP fluorescent gene, which proves that GDF5 and anti-MMP9 nanoantibodies were successfully modified onto mesenchymal stem cells.

[0039] 3.2IL-10 is an anti-inflammatory factor, FOXP3 is a key regulatory T cell specific transcription factor, and high expression of FOXP3 is closely related to arthritis. Therefore, the therapeutic effect of mesenchymal stem cells on arthritis can be directly evaluated by verifying the expression of anti-inflammatory factors and proteins closely related to arthritis. According to the experimental results, after stem cell treatment, the unmodified mesenchymal stem cell treatment group, the mesenchymal stem cell treatment group of comparative example 1, the mesenchymal stem cell treatment group of comparative example 2, and the mesenchymal stem cell treatment group of Example 1 expressed the anti-inflammatory factor IL-10 more than the PBS control group, and the expression amount gradually increased; while the FOXP3 protein closely related to arthritis, the unmodified mesenchymal stem cell treatment group, the mesenchymal stem cell treatment group of comparative example 1, the mesenchymal stem cell treatment group of comparative example 2, and the mesenchymal stem cell treatment group of Example 1 were expressed less than the PBS control group, and the expression amount gradually decreased. It can be seen that after stem cell treatment, the inflammatory response of rat arthritis was greatly reduced, and arthritis was effectively treated. Among them, the therapeutic effect of the double-modified mesenchymal stem cell treatment group was significantly higher than that of the single-modified mesenchymal stem cell treatment group, and the therapeutic effect of the single-modified mesenchymal stem cell treatment group was higher than that of the unmodified mesenchymal stem cells. Therefore, mesenchymal stem cells double-modified with GDF5 and anti-MMP9 nanoantibodies have a good effect in treating arthritis.

[0040] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A mesenchymal stem cell, characterized in that: The mesenchymal stem cells are obtained by double modification of the GDF5 gene and anti-MMP9 nanoantibody.

2. The mesenchymal stem cell according to claim 1, characterized in that The nucleotide sequence of the GDF5 gene is shown in SEQ ID No.

3.

3. The mesenchymal stem cell according to claim 1, characterized in that The nucleotide sequence of the anti-MMP9 nanobody is shown in SEQ ID No.

4.

4. The mesenchymal stem cell according to claim 1, characterized in that The mesenchymal stem cells are derived from placenta, umbilical cord or bone marrow tissue.

5. A method for preparing mesenchymal stem cells according to claim 1, characterized in that: The method comprises the following steps: S1. Preparation of mesenchymal stem cells: Take placenta, umbilical cord or bone marrow tissue containing primary mesenchymal stem cells, cut small pieces, wash with phosphate buffer on a culture dish until the washing solution is transparent, and then cut into pieces; transfer the cut tissue to a 50ml centrifuge tube, add 25ml collagenase IV with a mass / volume concentration of 0.1%, and shake and digest in a 37°C constant temperature incubator for 30min; add 20ml phosphate buffer to the digested tissue, mix well and filter through a 100μm mesh, centrifuge the filtrate at 1200 rpm in a 4°C centrifuge for 5min, remove the supernatant and retain the cell pellet; add 5ml of DMEM culture medium containing 20% ​​fetal bovine serum to the cell pellet, mix well and inoculate into a cell bottle, culture at 37°C, 5% CO2, change the medium every 2 days, and pass it on; S2. Construct lentiviral vectors; directly synthesize GDF5 and anti-MMP9 nanoantibody nucleotide sequences, connect them into the restriction-cut lentiviral expression vector GV367 (AgeI / NheI restriction, element sequence Ubi-MCS-SV40-EGFP-IRES-puromycin), construct recombinant plasmids, and transform DH5α competent cells for amplification. Extract plasmids for sequencing verification, and save the successfully constructed plasmids; digest 293T cells in the logarithmic phase to make a cell suspension and inoculate them on a 24-well culture plate, culture them in a cell culture incubator until the cell confluence rate is about 70-80%, and co-transfect 293T cells with plasmid DNA, packaging plasmid pHelper1.0 vector, envelope plasmid pHelper2.0 vector and LIPOFECTAMINE 3000. After 6 hours of culture, replace with fresh culture medium, continue to culture for 72 hours, collect the lentivirus-rich 293T cell supernatant, filter, centrifuge, and concentrate to obtain a high-titer virus concentrate; S3, take MSCs within 5 generations in the logarithmic growth phase, inoculate them in 12 culture plates at 100,000 cells / well, 1 ml of culture medium per well. When the cell confluency is about 50%, remove the culture medium, wash with PBS and transfect the virus concentrate in step S2, the transfection time is 10h. Then change the culture medium and continue to culture, and pass it according to the conventional method. Use the culture medium containing puromycin for screening for 7 days, change the medium every 2 days, and obtain MSCs that stably express GDF5 and anti-MMP9 nanoantibodies.

6. The preparation method according to claim 5, characterized in that: The amino acid sequence of GDF5 is shown in SEQ ID No.1, and the nucleotide sequence is shown in SEQ ID No.

3.

7. The preparation method according to claim 5, characterized in that: The amino acid sequence of the anti-MMP9 nanobody is shown in SEQ ID No.2, and the nucleotide sequence is shown in SEQ ID No.

4.

8. The use of mesenchymal stem cells according to claim 1, characterized in that: The application is to prepare medicine for treating arthritis.

9. The use according to claim 8, characterized in that: The arthritis is caused by autoimmune reaction, infection, metabolic disorder, trauma, degenerative disease.

10. The use according to claim 8, characterized in that: The arthritis is rheumatoid arthritis, rheumatic arthritis or osteoarthritis.

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