Mesenchymal Stem Cell Injection, Preparation Process and Application in the Treatment of Autoimmune Diseases

Abstract

The present invention provides a mesenchymal stem cell injection solution, a preparation process and an application in the treatment of autoimmune diseases, belonging to the technical field of biomedicine. The mesenchymal stem cell injection solution provided by the present invention is prepared by using complete culture medium - 1, complete culture medium - 2, and complete culture medium - 3 under specific culture conditions. The mesenchymal stem cell working bank cells used in the present invention for preparing the mesenchymal stem cell injection solution have good cell safety, have good cell proliferation ability, highly express CD73, CD90, CD105, do not express CD19, CD34, CD45, CD11b, HLA - DR, have osteogenic and adipogenic differentiation abilities and good biological efficacy. The mesenchymal stem cell injection solution provided by the present invention meets the pharmacopoeia standards, has high contents of HGF and sTNFR1, and can play the effect of preventing and treating autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a mesenchymal stem cell injection solution, a preparation process and an application in the treatment of autoimmune diseases. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are widely sourced, easy to obtain, non-invasive to donors, and have unique immunomodulatory functions. They can inhibit the functions of various immune cells, such as T cells, B cells, NK cells, neutrophils, mononuclear macrophages, antigen-presenting cells, etc. Numerous in vivo or in vitro studies have shown that mesenchymal stem cells exhibit a strong inhibitory effect on the immune system, and thus are widely used in the clinical treatment of immune-related diseases, such as graft-versus-host disease, rheumatoid arthritis, etc.

[0003] Basic fibroblast growth factor (bFGF) can promote the growth, proliferation of stem cells and maintain the stemness of stem cells; stromal cell-derived factor-1α (SDF-1α) has the effects of promoting stem cell homing, chemotaxis and proliferation, and can accelerate the separation and acquisition of stem cells from tissues such as umbilical cords and is beneficial to maintaining the activity of stem cells when used in the early stage of mesenchymal stem cell isolation and culture; folic acid (vitamin B9) plays an important role in cell DNA synthesis and cell division; while methyl-β-cyclodextrin (MβCD) can improve the fluidity and integrity of cell membranes, improve cell culture efficiency, and regulate the cholesterol level on cell membranes in cell culture.

[0004] Tumor necrosis factor (TNF-α) is a pleiotropic cytokine. Biological and medical research has proven that TNF-α is involved in physiological and pathological processes such as immune regulation, inflammation, septic shock, apoptosis and autoimmunity, and plays an important role in maintaining the body's homeostasis and the occurrence and development of diseases. The interaction between TNF-α and two types of TNF receptors (TNFR1 and TNFR2) and the abnormal activation of downstream signaling pathways are important factors in the occurrence and development of autoimmune diseases such as rheumatoid arthritis and ulcerative colitis. Generally speaking, the inflammatory, apoptotic or programmed necrosis reactions caused by TNF-α are mainly triggered by binding to TNFR1. Soluble tumor necrosis factor type I receptor (sTNFR1) is the extracellular region of TNFR1, which can competitively block the binding of TNFR1 to TNF-α and reduce the toxicity of TNF-α to cells. Therefore, sTNFR1 can be used as an antagonist of TNF-α for the treatment of diseases and has important research value in the medical field.

[0005] c-Met has the activity of tyrosine kinase and mediates cell information transmission after being activated by ligands. It is an important regulatory factor for cell proliferation, differentiation and motility. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that can induce a variety of cellular responses, including proliferation, migration and survival, by activating its receptor c-met. Existing studies have shown that the HGF / c-met signal transduction pathway has a pro-inflammatory effect and can directly affect leukocyte responses in inflammation. Therefore, targeted inhibition of the HGF / c-met pathway may be a potential treatment method for immune-related diseases.

[0006] Currently, the research on umbilical cord mesenchymal stem cells in the clinical treatment of immune-related diseases has achieved initial results, but a more effective mesenchymal stem cell injection for the treatment of immune-related diseases needs to be developed. Summary of the Invention

[0007] Based on the problems and deficiencies in the prior art, the present invention aims to provide a mesenchymal stem cell injection, a preparation process and its application in the treatment of autoimmune diseases. The mesenchymal stem cell injection provided by the present invention is prepared under specific culture conditions using Complete Culture Medium - 1, Complete Culture Medium - 2, and Complete Culture Medium - 3; the Complete Culture Medium - 1 includes: a basal medium containing FBS, bFGF, SDF-1α and folic acid; the Complete Culture Medium - 2 includes: a basal medium containing FBS, bFGF, SDF-1α, folic acid and hydrogen; the Complete Culture Medium - 3 includes: a basal medium containing FBS, bFGF, methyl-β-cyclodextrin and folic acid. The mesenchymal stem cell working bank cells used in the present invention for preparing the mesenchymal stem cell injection have good cell safety, have good cell proliferation ability, highly express CD73, CD90, CD105, do not express CD19, CD34, CD45, CD11b, HLA-DR, have the differentiation ability of osteogenesis and adipogenesis, and have good biological efficacy. The mesenchymal stem cell injection provided by the present invention meets the pharmacopoeia standards, has high contents of HGF and sTNFR1, and can play the role of preventing and treating autoimmune diseases.

[0008] The technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a mesenchymal stem cell injection, and the preparation method of the mesenchymal stem cell injection includes the following steps:

[0010] S1. After the umbilical cord tissue is incubated in a culture flask, add Complete Culture Medium - 1 for culture and passage to obtain P0 generation human umbilical cord mesenchymal stem cells;

[0011] S2. Culture and passage the P0 generation human umbilical cord mesenchymal stem cells with Complete Culture Medium - 2 to obtain P1 generation human umbilical cord mesenchymal stem cells;

[0012] S3. Passage and culture the P1 generation of human umbilical cord mesenchymal stem cells with Complete Culture Medium - 3 to obtain the P2 generation of human umbilical cord mesenchymal stem cells. After centrifugation, discard the supernatant and collect the cells as seed bank cells;

[0013] S4. Continue to culture the seed bank cells with Complete Culture Medium - 3 until the P5 generation and cryopreserve them to obtain the working bank cells of mesenchymal stem cells;

[0014] S5. Resuspend the working bank cells of mesenchymal stem cells with cell preservation solution to obtain the mesenchymal stem cell injection.

[0015] Specifically, the Complete Culture Medium - 1 described in step S1 includes: a basal culture medium containing FBS, bFGF, SDF - 1α, and folic acid.

[0016] Preferably, the Complete Culture Medium - 1 described in step S1 includes: a basal culture medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 10 - 100 μg / L SDF - 1α, and 0.35 - 0.80 mg / L folic acid.

[0017] More preferably, the Complete Culture Medium - 1 described in step S1 includes: a basal culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF - 1α, and 0.35 mg / L folic acid.

[0018] Specifically, the Complete Culture Medium - 2 described in step S2 includes: a basal culture medium containing FBS, bFGF, SDF - 1α, folic acid, and hydrogen.

[0019] Preferably, the Complete Culture Medium - 2 described in step S2 includes: a basal culture medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 10 - 100 μg / L SDF - 1α, 0.35 - 0.80 mg / L folic acid, and 0.5 - 2 mg / L hydrogen.

[0020] More preferably, the Complete Culture Medium - 2 described in step S2 includes: a basal culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF - 1α, 0.35 mg / L folic acid, and 1 mg / L hydrogen.

[0021] Specifically, the Complete Culture Medium - 3 described in steps S3 and S4 includes: a basal culture medium containing FBS, bFGF, methyl - β - cyclodextrin, and folic acid.

[0022] Preferably, the complete culture medium - 3 described in steps S3 and S4 comprises a basal culture medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 1 - 2 mg / L methyl - β - cyclodextrin, and 0.35 - 0.80 mg / L folic acid.

[0023] More preferably, the complete culture medium - 3 described in steps S3 and S4 comprises a basal culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 1 mg / L methyl - β - cyclodextrin, and 0.35 mg / L folic acid.

[0024] Preferably, the basal culture medium includes but is not limited to DMEM / F12 culture medium, DMEM culture medium, MSC growth medium, RPMI 1640, or E8.

[0025] More preferably, the basal culture medium is DMEM / F12 culture medium.

[0026] Specifically, the culture conditions in step S1 include 5% CO2 and 1% - 2% O2.

[0027] Preferably, the culture conditions in step S1 include 5% CO2 and 1% O2.

[0028] Specifically, step S1 includes: After the umbilical cord tissue is incubated in a culture flask with 5% CO2 for 24 h, complete culture medium - 1 is added and cultured and passaged under the conditions of 5% CO2 and 1% - 2% O2 to obtain passage 0 human umbilical cord mesenchymal stem cells.

[0029] Preferably, step S1 includes: After the umbilical cord tissue is incubated in a culture flask with 5% CO2 for 24 h, complete culture medium - 1 is added and cultured and passaged under the conditions of 5% CO2 and 1% O2 to obtain passage 0 human umbilical cord mesenchymal stem cells.

[0030] Specifically, the culture in step S1 includes: 5 mL of complete culture medium - 1 is added, and after overnight culture, 10 mL of complete culture medium - 1 is supplemented and the culture is continued.

[0031] Specifically, the culture conditions in step S2 include 5% CO2 and 5% - 10% O2;

[0032] Preferably, the culture conditions in step S2 include 5% CO2 and 5% O2.

[0033] Specifically, the culture conditions in step S3 include 5% CO2 and 18% - 20% O2.

[0034] Preferably, the culture conditions in step S3 include 5% CO2 and 18% O2.

[0035] Specifically, the cell preservation solution described in step S5 is composed of human albumin, heparin, dextran - 40, and compound electrolyte solution.

[0036] Preferably, the cell preservation solution described in step S5 includes 10 - 20 mg / mL of human albumin, 50 - 100 IU / mL of heparin, and 50 - 100 mg / mL of dextran - 40.

[0037] More preferably, the cell preservation solution described in step S5 includes 10 mg / mL of human albumin, 50 IU / mL of heparin, and 60 mg / mL of dextran - 40.

[0038] Specifically, the density of mesenchymal stem cells in the mesenchymal stem cell injection solution is 0.5×10 6 -1×10 8 cells / mL.

[0039] Preferably, the density of mesenchymal stem cells in the mesenchymal stem cell injection solution is 1×10 6 cells / mL.

[0040] On the other hand, the present invention provides a method for preparing the above - mentioned mesenchymal stem cell injection solution.

[0041] Specifically, the preparation method includes the following steps:

[0042] S1. After the umbilical cord tissue is placed in a culture flask and incubated, complete culture medium - 1 is added for sub - culture to obtain P0 - generation human umbilical cord mesenchymal stem cells;

[0043] S2. The P0 - generation human umbilical cord mesenchymal stem cells are cultured and sub - cultured with complete culture medium - 2 to obtain P1 - generation human umbilical cord mesenchymal stem cells;

[0044] S3. The P1 - generation human umbilical cord mesenchymal stem cells are cultured and sub - cultured with complete culture medium - 3 to obtain P2 - generation human umbilical cord mesenchymal stem cells. After centrifugation, the supernatant is discarded, and the cells are collected as seed bank cells;

[0045] S4. The seed bank cells are continuously cultured with complete culture medium - 3 until P5 - generation and cryopreserved to obtain mesenchymal stem cell working bank cells;

[0046] S5. The mesenchymal stem cell working bank cells are resuspended with the cell preservation solution to obtain the mesenchymal stem cell injection solution.

[0047] On another aspect, the present invention provides a method for identifying the cell activity of the above - mentioned mesenchymal stem cell injection solution. The cell activity identification method includes detecting the cell morphology, cell viability, flow cytometry phenotype, osteogenic and adipogenic differentiation ability, or biological potency of the mesenchymal stem cells.

[0048] Specifically, the detection of the flow cytometry phenotype includes detecting one or more of CD73, CD90, CD105, CD19, CD34, CD45, CD11b, and HLA-DR.

[0049] Preferably, the detection criteria for the flow cytometry phenotype are high expression of CD73, CD90, and CD105, and no expression of CD19, CD34, CD45, CD11b, and HLA-DR.

[0050] Specifically, the detection of the biological potency includes detecting HGF or sTNFR1.

[0051] Preferably, the physiological range of HGF is 10 - 30 ng / 1E+6 cells.

[0052] Preferably, the physiological range of sTNFR1 is 220 - 800 pg / 1E+6 cells.

[0053] In another aspect, the present invention provides the use of the above-mentioned mesenchymal stem cell injection solution, the mesenchymal stem cell injection solution prepared by the preparation method, or the mesenchymal stem cell injection solution meeting the standards of the cell activity identification method in the preparation of a drug containing mesenchymal stem cells.

[0054] Specifically, the drug includes a drug for preventing or treating autoimmune diseases.

[0055] Preferably, the autoimmune diseases include but are not limited to systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, idiopathic thrombocytopenic purpura, or autoimmune hemolytic anemia.

[0056] More preferably, the autoimmune diseases are rheumatoid arthritis and systemic lupus erythematosus.

[0057] The beneficial effects of the present invention are as follows:

[0058] (1) The mesenchymal stem cell working bank cells used in the present invention to prepare the mesenchymal stem cell injection solution have good cell safety, have good cell proliferation ability, highly express CD73, CD90, CD105, do not express CD19, CD34, CD45, CD11b, HLA-DR, have osteogenic and adipogenic differentiation abilities, and have good biological potency.

[0059] (2) The mesenchymal stem cell injection solution provided by the present invention meets the standards of the Pharmacopoeia of the People's Republic of China, has high contents of HGF and sTNFR1, and can play the role of preventing and treating autoimmune diseases. Description of the Drawings

[0060] Figure 1 is the cell proliferation ability.

[0061] Figure 2 is the cell differentiation ability.

[0062] Figure 3 is the joint swelling score of CIA mice. *** in the figure represents P <0.001, # represents P <0.05.

[0063] Figure 4 is the IFN-γ level in the serum of CIA mice. ** in the figure represents P <0.01, # represents P <0.05.

[0064] Figure 5 is the IL-6 level in the serum of CIA mice. ** in the figure represents P <0.01, ## represents P <0.01.

[0065] Figure 6 is the TNF-α level in the serum of CIA mice. ** in the figure represents P <0.01, # represents P <0.05.

[0066] Figure 7 is the clinical observation score of SLE mice. ** in the figure represents P <0.01, # represents P <0.05.

[0067] Figure 8 is the detection result of anti-double-stranded DNA IgG in SLE mice. *** in the figure represents P <0.001, ## represents P <0.01.

[0068] Figure 9 is the clinical score of psoriasis mice. # in the figure represents P <0.05.

[0069] Figure 10 is the skin thickness result of psoriasis mice. ** in the figure represents P <0.01, ## represents P <0.01. Specific implementation manners

[0070] The present invention will be described below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention, so that the technical solutions of the present invention can be more easily understood and grasped. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0071] The experimental materials used in the present invention are shown in Table 1:

[0072] Table 1 Experimental materials

[0073]

[0074] Example 1 Preparation and detection of mesenchymal stem cell working bank cells

[0075] 1. Preparation of mesenchymal stem cell working bank cells

[0076] 1.1 Preparation of mesenchymal stem cell working bank cell 1

[0077] S1. Human umbilical cord tissue is derived from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue longer than 15 cm, cut the umbilical cord tissue into small pieces with a diameter of 1-1.5 mm, without washing, so as to retain the original environment of the tissue; then directly spread each piece of umbilical cord tissue block on a T75 culture flask, and incubate it in a 5% CO2 incubator at 38 °C for 24 hours, without adding cell culture medium during this process;

[0078] After incubation, place the culture flask containing umbilical cord tissue in a 5% CO2, 1% O2 incubator at 38 °C, add 5 mL of complete culture medium -1 to the culture flask, supplement 10 mL of complete culture medium -1 after overnight culture, and then change the culture medium every 3-5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain passage 0 human umbilical cord mesenchymal stem cells.

[0079] S2. Centrifuge the passage 0 human umbilical cord mesenchymal stem cells obtained in step S1 at 300-400 g for 5 min, discard the supernatant, add complete culture medium -2, and place it in a 5% CO2, 5% O2 environment incubator at 37 °C for subculture for 48-72 hours, denoted as passage 1.

[0080] S3. Centrifuge the passage 1 human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g × 5 min, discard the supernatant, add complete culture medium -3, and place it in a 5% CO2, 18% O2 environment incubator at 37 °C for subculture for 48-72 hours, denoted as passage 2. The obtained passage 2 human umbilical cord mesenchymal stem cells are centrifuged at 400 g × 5 min, discard the supernatant, and collect the cells as the seed bank.

[0081] S4. Take the seed bank cells described in step S3, continue to culture them in complete culture medium -3 until the P5 generation, and then cryopreserve them to obtain mesenchymal stem cell working bank cells (denoted as W1). The culture system and environment are as follows: complete culture medium -3, 37°C, 5% CO2.

[0082] The complete culture medium -1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, and 0.35 mg / L folic acid;

[0083] The complete culture medium -2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, 0.35 mg / L folic acid, and 1 mg / L hydrogen;

[0084] The complete culture medium -3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 1 mg / L methyl-β-cyclodextrin, and 0.35 mg / L folic acid.

[0085] 1.2 Preparation of mesenchymal stem cell working bank cells 2

[0086] S1. The human umbilical cord tissue is from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue longer than 15 cm, cut the umbilical cord tissue into small segments with a diameter of 1 - 1.5 mm without washing to retain the original tissue environment; then directly spread each piece of umbilical cord tissue on a T75 culture flask and incubate it in a 5% CO2 incubator at 38°C for 24 hours without adding cell culture medium.

[0087] After incubation, place the culture flask containing the umbilical cord tissue in a 5% CO2, 2% O2 incubator at 38°C, add 5 mL of complete culture medium -1 to the culture flask, after overnight culture, supplement 10 mL of complete culture medium -1, and thereafter, change the culture medium every 3 - 5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0088] S2. Centrifuge the P0 generation human umbilical cord mesenchymal stem cells obtained in step S1 at 300 - 400 g for 5 min, discard the supernatant, add complete culture medium -2, and place it in a culture incubator at 37°C in a 5% CO2, 10% O2 environment for subculture for 48 - 72 hours, denoted as the P1 generation.

[0089] S3. Centrifuge the P1 generation of human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g for 5 min, discard the supernatant, add complete culture medium - 3, and place it in an incubator at 37 °C in an environment of 5% CO2 and 20% O2 for subculture for 48 - 72 hours, denoted as the P2 generation. Centrifuge the obtained P2 generation of human umbilical cord mesenchymal stem cells at 400 g for 5 min, discard the supernatant, and collect the cells as the seed bank.

[0090] S4. Take the cells from the seed bank described in step S3, continue to culture them with complete culture medium - 3 until the P5 generation and cryopreserve them to obtain mesenchymal stem cell working bank cells (denoted as W2). The culture system and environment are: complete culture medium - 3, 37 °C, 5% CO2.

[0091] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 15% v / v FBS, 10 μg / L bFGF, 10 μg / L SDF - 1α, and 0.8 mg / L folic acid;

[0092] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 15% v / v FBS, 10 μg / L bFGF, 10 μg / L SDF - 1α, 0.8 mg / L folic acid, and 0.5 mg / L hydrogen;

[0093] The complete culture medium - 3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 15% v / v FBS, 10 μg / L bFGF, 1.5 mg / L methyl - β - cyclodextrin, and 0.8 mg / L folic acid.

[0094] 1.3 Preparation of mesenchymal stem cell working bank cells 3

[0095] S1. Human umbilical cord tissue is derived from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue over 15 cm, cut the umbilical cord tissue into small segments with a diameter of 1 - 1.5 mm without washing to retain the original tissue environment; then directly spread each piece of umbilical cord tissue block on a T75 culture flask and incubate it in a 5% CO2 incubator at 38 °C for 24 hours without adding cell culture medium;

[0096] After incubation, place the culture flask containing umbilical cord tissue in a 5% CO2 and 1% O2 incubator at 38 °C, add 5 mL of complete culture medium - 1 to the culture flask, add 10 mL of complete culture medium - 1 after overnight culture, and thereafter, change the culture medium every 3 - 5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain P0 generation of human umbilical cord mesenchymal stem cells.

[0097] S2. Centrifuge the P0 generation of human umbilical cord mesenchymal stem cells obtained in step S1 at 300 - 400 g for 5 min, discard the supernatant, add complete culture medium - 2, and place it in an incubator at 37°C in an environment of 5% CO2 and 7.5% O2 for subculture for 48 - 72 hours, denoted as the P1 generation.

[0098] S3. Centrifuge the P1 generation of human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g × 5 min, discard the supernatant, add complete culture medium - 3, and place it in an incubator at 37°C in an environment of 5% CO2 and 18% O2 for subculture for 48 - 72 hours, denoted as the P2 generation. For the obtained P2 generation of human umbilical cord mesenchymal stem cells, centrifuge at 400 g × 5 min, discard the supernatant, and collect the cells as the seed bank.

[0099] S4. Take the cells from the seed bank described in step S3, continue to culture them with complete culture medium - 3 until the P5 generation and cryopreserve them to obtain the mesenchymal stem cell working bank cells (denoted as W3). The culture system and environment are: complete culture medium - 3, 37°C, 5% CO2.

[0100] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 12.5% v / v FBS, 5 μg / L bFGF, 100 μg / L SDF - 1α, and 0.4 mg / L folic acid;

[0101] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 12.5% v / v FBS, 5 μg / L bFGF, 100 μg / L SDF - 1α, 0.4 mg / L folic acid, and 2 mg / L hydrogen;

[0102] The complete culture medium - 3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 12.5% v / v FBS, 5 μg / L bFGF, 2 mg / L methyl - β - cyclodextrin, and 0.4 mg / L folic acid.

[0103] 1.4 Preparation of mesenchymal stem cell working bank cells 4

[0104] The difference between mesenchymal stem cell working bank cells 4 (W4) and mesenchymal stem cell working bank cells 1 (W1) is only that: the complete culture medium - 1, complete culture medium - 2, and complete culture medium - 3 are different.

[0105] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF;

[0106] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF;

[0107] The complete culture medium - 3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF.

[0108] 1.5 Preparation of mesenchymal stem cell working bank cell 5

[0109] The difference between mesenchymal stem cell working bank cell 5 (W5) and mesenchymal stem cell working bank cell 1 (W1) is only that: the complete culture medium - 1, complete culture medium - 2, and complete culture medium - 3 are different.

[0110] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, and 50 μg / L SDF - 1α;

[0111] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF - 1α, and 1 mg / L hydrogen;

[0112] The complete culture medium - 3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, and 1 mg / L methyl - β - cyclodextrin.

[0113] 1.6 Preparation of mesenchymal stem cell working bank cell 6

[0114] The difference between mesenchymal stem cell working bank cell 6 (W6) and mesenchymal stem cell working bank cell 1 (W1) is only that: the complete culture medium - 1 and complete culture medium - 2 are different.

[0115] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, and 0.35 mg / L folic acid;

[0116] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 0.35 mg / L folic acid, and 1 mg / L hydrogen;

[0117] 1.7 Preparation of mesenchymal stem cell working bank cell 7

[0118] The difference between mesenchymal stem cell working bank cell 7 (W7) and mesenchymal stem cell working bank cell 1 (W1) lies only in that the complete culture medium - 3 is different.

[0119] The complete culture medium - 3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, and 0.35 mg / L folic acid.

[0120] 1.8 Preparation of mesenchymal stem cell working bank cell 8

[0121] S1. Human umbilical cord tissue is derived from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue over 15 cm, cut the umbilical cord tissue into small pieces with a diameter of 1 - 1.5 mm without washing to retain the original tissue environment; then directly spread each piece of umbilical cord tissue block onto a T75 culture flask and incubate it in a 5% CO2 incubator at 38 °C for 24 hours without adding cell culture medium.

[0122] After incubation, place the culture flask containing umbilical cord tissue in a 5% CO2 incubator at 38 °C, add 5 mL of complete culture medium - 1 to the culture flask, supplement 10 mL of complete culture medium - 1 after overnight culture, and then change the culture medium every 3 - 5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain passage 0 human umbilical cord mesenchymal stem cells.

[0123] S2. Centrifuge the passage 0 human umbilical cord mesenchymal stem cells obtained in step S1 at 300 - 400 g for 5 min, discard the supernatant, add complete culture medium - 2, and perform subculture in a 5% CO2 environment incubator at 37 °C for 48 - 72 hours, denoted as passage 1.

[0124] S3. Centrifuge the passage 1 human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g × 5 min, discard the supernatant, add complete culture medium - 3, and perform subculture in a 5% CO2 environment incubator at 37 °C for 48 - 72 hours, denoted as passage 2. Centrifuge the obtained passage 2 human umbilical cord mesenchymal stem cells at 400 g × 5 min, discard the supernatant, and collect the cells as the seed bank.

[0125] S4. Take the cells from the seed bank described in step S3, continue to culture them to passage 5 with complete culture medium - 3 and cryopreserve them to obtain mesenchymal stem cell working bank cell (W8). The culture system and environment are: complete culture medium - 3, 37 °C, 5% CO2.

[0126] The complete culture medium - 1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, and 0.35 mg / L folic acid;

[0127] The complete culture medium - 2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, 0.35 mg / L folic acid, and 1 mg / L hydrogen;

[0128] The complete culture medium - 3 described in step S3 and step S4 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 1 mg / L methyl-β-cyclodextrin, and 0.35 mg / L folic acid.

[0129] 1.9 Preparation of mesenchymal stem cell working bank cell 9

[0130] S1. Human umbilical cord tissue is derived from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue over 15 cm, cut the umbilical cord tissue into small segments with a diameter of 1 - 1.5 mm without washing to retain the original tissue environment; then directly spread and inoculate each umbilical cord tissue block into a T75 culture flask, and incubate it in a 5% CO2 incubator at 38 °C for 24 hours without adding cell culture medium;

[0131] After incubation, place the culture flask containing umbilical cord tissue in a 5% CO2, 5% O2 incubator at 38 °C, add 5 mL of complete culture medium - 1 to the culture flask, supplement 10 mL of complete culture medium - 1 after overnight culture, and thereafter, change the culture medium every 3 - 5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0132] S2. Centrifuge the P0 generation human umbilical cord mesenchymal stem cells obtained in step S1 at 300 - 400 g for 5 min, discard the supernatant, add complete culture medium - 2, and place it in an incubator at 37 °C in an environment of 5% CO2 and 15% O2 for subculture for 48 - 72 hours, denoted as P1 generation.

[0133] S3. Centrifuge the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g × 5 min, discard the supernatant, add complete culture medium - 3, and place it in an incubator at 37 °C in an environment of 5% CO2 and 20% O2 for subculture for 48 - 72 hours, denoted as P2 generation. Centrifuge the obtained P2 generation human umbilical cord mesenchymal stem cells at 400 g × 5 min, discard the supernatant, and collect the cells as the seed bank.

[0134] S4. Take the seed bank cells described in step S3, continue to culture them in complete culture medium -3 until the P5 generation and then cryopreserve them to obtain mesenchymal stem cell working bank cells (W9). The culture system and environment are: complete culture medium -3, 37 °C, 5% CO2.

[0135] The complete culture medium -1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, and 0.35 mg / L folic acid;

[0136] The complete culture medium -2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF-1α, 0.35 mg / L folic acid, and 1 mg / L hydrogen;

[0137] The complete culture medium -3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS, 7.5 μg / L bFGF, 1 mg / L methyl-β-cyclodextrin, and 0.35 mg / L folic acid.

[0138] 1.10 Preparation of mesenchymal stem cell working bank cells 10

[0139] S1. The human umbilical cord tissue is from healthy fetuses born by cesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissues longer than 15 cm, cut the umbilical cord tissues into small segments with a diameter of 1 - 1.5 mm without washing to retain the original tissue environment; then directly spread each umbilical cord tissue block on a T75 culture flask and incubate it in a 5% CO2 incubator at a temperature of 38 °C for 24 hours without adding cell culture medium;

[0140] After incubation, place the culture flask containing the umbilical cord tissue in a 5% CO2 incubator at 38 °C, add 5 mL of complete culture medium -1 to the culture flask, supplement 10 mL of complete culture medium -1 after overnight culture, and thereafter, change the culture medium every 3 - 5 days. After the cell confluence reaches 80%, digest it with trypsin digestion solution to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0141] S2. Centrifuge the P0 generation human umbilical cord mesenchymal stem cells obtained in step S1 at 300 - 400 g for 5 min, discard the supernatant, add complete culture medium -2, and perform subculture in a culture flask in a 5% CO2 environment at 37 °C for 48 - 72 hours, denoted as the P1 generation.

[0142] S3. Centrifuge the P1 generation of human umbilical cord mesenchymal stem cells obtained in step S2 at 400 g for 5 min, discard the supernatant, add complete culture medium -3, and perform subculture at 37 °C in an incubator with a 5% CO2 environment for 48 - 72 hours, denoted as the P2 generation. For the obtained P2 generation of human umbilical cord mesenchymal stem cells, centrifuge at 400 g for 5 min, discard the supernatant, and collect the cells as the seed bank.

[0143] S4. Take the cells from the seed bank described in step S3, continue to culture them to the P5 generation with complete culture medium -3 and then cryopreserve them to obtain mesenchymal stem cell working bank cells (W10). The culture system and environment are: complete culture medium -3, 37 °C, 5% CO2.

[0144] The complete culture medium -1 described in step S1 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF;

[0145] The complete culture medium -2 described in step S2 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF;

[0146] The complete culture medium -3 described in steps S3 and S4 is: DMEM / F12 culture medium containing 10% v / v FBS and 7.5 μg / L bFGF.

[0147] 2. Detection of mesenchymal stem cell working bank cells

[0148] Take the mesenchymal stem cell working bank cells 1 - mesenchymal stem cell working bank cells 10 prepared above for detection of bacterial, mycoplasma, and virus safety; detection of cell morphology, cell proliferation, flow cytometry phenotype, osteogenic and adipogenic differentiation ability, and biological potency (HGF, sTNFR1).

[0149] 2.1 Sterility, mycoplasma, and virus safety detection

[0150] Perform sterility detection according to the sterility test method in General Chapter 1101, Part III of the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0151] Perform mycoplasma detection according to the first method in General Chapter 3301, Part III of the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0152] According to the kit instructions, use the PCR method to complete the detection of viruses such as EBV, HCMV, and HIV in the working bank cells.

[0153] The determination results show that the bacteria, mycoplasma, and viruses in mesenchymal stem cell working bank cells 1 - mesenchymal stem cell working bank cells 10 are all negative.

[0154] 2.2 Cell morphology and cell proliferation assay

[0155] The cell status of mesenchymal stem cell working bank cells 1 (W1) - mesenchymal stem cell working bank cells 10 (W10) was observed using an inverted microscope. The measurement results showed that the cell status of W1 - W10 was good, with individual cells being spindle-shaped and growing in a fusiform shape when fused.

[0156] Take mesenchymal stem cell working bank cells 1 (W1) - mesenchymal stem cell working bank cells 10 (W10), inoculate 10,000 cells per well into a 96-well plate, and analyze and measure cell proliferation using the CCK-8 method at 0 h, 24 h, 48 h, 72 h, and 96 h after inoculation. The measurement results are as Figure 1 shown, among which the cell proliferation ability of W1 - W3 is the strongest, that of W4 - W9 is relatively poor, and that of W10 is the worst.

[0157] 2.3 Flow cytometry phenotype detection

[0158] Take mesenchymal stem cell working bank cells 1 - mesenchymal stem cell working bank cells 10 and perform the following operations: Collect 5×10 6 cells, evenly distribute them into 10 centrifuge tubes, centrifuge at 300 g for 5 minutes, then aspirate and discard the supernatant, and add the following antibodies respectively: PE-IgG1, PE-CD73, PE CD90, PE-CD105, PE-CD45, PE-HLA-DR, PE-CD11b, FITC-IgG1, FITC-CD19, and FITC-CD34 (antibodies from BD Biosciences, USA), and detect cell surface antigens using a BD Calibur flow cytometer.

[0159] The measurement results are shown in Table 2: Mesenchymal stem cell working bank cells (W1 - W10) all highly express CD73, CD90, and CD105, and do not express CD19, CD34, CD45, CD11b, and HLA-DR.

[0160] Table 2 Flow cytometry phenotypes of mesenchymal stem cell working bank cells 1 - 10 (W1 - W10)

[0161]

[0162] 2.4 Detection of osteogenic and adipogenic differentiation abilities

[0163] Take mesenchymal stem cell working bank cells 1 - mesenchymal stem cell working bank cells 10 and perform the following operations:

[0164] Each group of cells with *10 4Inoculate the cells in a 224-well plate by holes. After the cell confluence reaches about 80%, change to the adipogenic and osteogenic induction medium, and change it every 2 - 3 days. On the 21st day of induction culture, fix the cells with 4% paraformaldehyde solution at room temperature for 30 minutes, then stain with 10% Oil Red-O and observe the adipogenic differentiation under a microscope, and stain with alizarin red solution and observe the osteogenic differentiation under a microscope. The measurement results are as Figure 2 shown. Each group of cells has the ability of osteogenic and adipogenic differentiation. Among them, the cells of W1 and W2 have the strongest osteogenic and adipogenic differentiation ability, followed by W3, W8, and W9, the differentiation ability of W4 - W7 is weaker, and the differentiation ability of W10 is the weakest.

[0165] 2.5 Biological potency detection

[0166] Take the mesenchymal stem cell working bank cells (W1 - W10), inoculate them into 6-well plates respectively. After culturing for 48 h, take the supernatant, and at the same time digest the cells for counting. Use ELISA method (product of R&D systems company) to detect the contents of HGF and sTNFR1 in the supernatant (Table 3).

[0167] It can be found through detection that the HGF and sTNFR1 secreted by the cells in the W1 group are the highest, followed by W2 and W3, lower in W4 - W6, and the lowest in W10.

[0168] Table 3 Contents of HGF and sTNFR1

[0169]

[0170] Example 2 Preparation and detection of mesenchymal stem cell injection

[0171] 1. Preparation of mesenchymal stem cell injection

[0172] 1.1 Preparation of mesenchymal stem cell injection 1

[0173] Resuscitate and culture the mesenchymal stem cell working bank cell 1 prepared in Example 1 until the cell confluence reaches 80%. Then digest with trypsin, terminate, centrifuge, and wash 3 times with compound electrolyte solution, and resuspend with compound electrolyte solution containing 10 mg / mL human albumin, 50 IU / mL heparin, and 60 mg / mL dextran - 40 to make mesenchymal stem cell injection 1 (P1). The volume of the injection is 20 mL, and the number of cells is 1E+7.

[0174] 1.2 Preparation of mesenchymal stem cell injection 2

[0175] After the mesenchymal stem cell working bank cells 2 prepared in Example 1 were thawed and cultured until 80% cell confluence, they were digested with trypsin, centrifuged, washed 3 times with compound electrolyte solution, and resuspended with a compound electrolyte solution containing 15 mg / mL human albumin, 75 IU / mL heparin and 50 mg / mL dextran-40 to prepare mesenchymal stem cell injection 2 (P2).

[0176] 1.3 Preparation of mesenchymal stem cell injection 3

[0177] After the mesenchymal stem cell working bank cells 3 prepared in Example 1 were thawed and cultured until 80% cell confluence, they were digested with trypsin, centrifuged, washed 3 times with compound electrolyte solution, and resuspended with a compound electrolyte solution containing 20 mg / mL human albumin, 100 IU / mL heparin and 100 mg / mL dextran-40 to prepare mesenchymal stem cell injection 3 (P3).

[0178] 2. Detection of mesenchymal stem cell injection

[0179] 2.1 Visual inspection for foreign particles

[0180] Visual inspection for foreign particles was performed according to the method for visual inspection of foreign particles in General Chapter 0904 of Part III of the Chinese Pharmacopoeia 2020 Edition.

[0181] 2.2 Inspection for insoluble particles

[0182] Inspection for insoluble particles was carried out according to the first method in General Chapter 0903 of Part III of the Chinese Pharmacopoeia 2020 Edition.

[0183] 2.3 Inspection for filling volume

[0184] Inspection for filling volume was performed according to General Chapter 0102 of Part III of the Chinese Pharmacopoeia 2020 Edition.

[0185] 2.4 Osmolality inspection

[0186] The osmolality was determined according to General Chapter 0632 of Part III of the Chinese Pharmacopoeia 2020 Edition.

[0187] 2.5 Cell number and cell viability

[0188] A cell counter was used for cell counting. The stem cell preparation to be tested was stained with 0.04% trypan blue (1:1), and the cell number was read and the viability was calculated.

[0189] 2.6 Sterility test

[0190] Sterility test was carried out according to the sterility test method in General Chapter 1101 of Part III of the Chinese Pharmacopoeia 2020 Edition.

[0191] 2.7 Mycoplasma Detection

[0192] Mycoplasma detection was performed according to the first method in General Chapter 3301, Part III of the Chinese Pharmacopoeia 2020 Edition.

[0193] 2.8 Endotoxin Detection

[0194] Endotoxin detection was performed according to the gel method in General Chapter 1143, Part III of the Chinese Pharmacopoeia 2020 Edition.

[0195] 2.9 Results of HGF and sTNFR1

[0196] The mesenchymal stem cell injection (P1-3) cells obtained in Example 2 were inoculated into T75 culture flasks at a density of 1×10 4 / cm 2 After culturing for 48 h, the supernatant was harvested, the cells were digested and counted, and the contents of HGF and sTNFR1 in the samples were detected according to the instructions of the HGF and sTNFR1 kits.

[0197] The pharmacopoeia standards for visible foreign matters, insoluble particles, filling volume difference, osmotic pressure, cell number, cell viability, sterility test, mycoplasma detection, and endotoxin detection are shown in Table 4. The detection results of mesenchymal stem cell injection (P1-P3) are shown in Table 5, and all the detections of mesenchymal stem cell injection are qualified. The detection results of HGF and sTNFR1 are shown in Table 6, and the detection results are found.

[0198] Table 4 Pharmacopoeia Standards

[0199]

[0200] Table 5 Detection Results of Mesenchymal Stem Cell Injection

[0201]

[0202] Table 6 Results of HGF and sTNFR1 in Mesenchymal Stem Cell Injection

[0203]

[0204] Example 3 Evaluation of the Effect of Mesenchymal Stem Cell Injection on CIA Mice

[0205] 1. Construction of CIA Model and Administration

[0206] In this example, 50 male DBA / 1 mice aged 6 - 8 weeks were used for the experiment. The DBA / 1 mice were randomly divided into 5 groups, namely the normal control group, the model group, and the mesenchymal stem cell injection groups (P1 - P3), with 10 mice in each group. The mice in the model group were modeled as follows: A bovine type II collagen solution with a concentration of 4 mg / mL was mixed with complete Freund's adjuvant in equal volume, and the injection emulsion obtained after sufficient emulsification was used for intradermal injection at the base of the tail. On the 1st day and the 21st day of the experiment, 100 μL of the injection emulsion was intradermally injected at the base of the tail of the mice in the model group and the mesenchymal stem cell injection groups. On the 1st day and the 21st day of the experiment, 100 μL of PBS solution was intradermally injected at the base of the tail of the mice in the normal control group.

[0207] On the 22nd day of the experiment, in the mesenchymal stem cell treatment group, mesenchymal stem cell injection (P1 - P3) was intravenously injected at 2x10 6 cells / mouse, and the normal control group and the model group were injected with an equal volume of normal saline via the tail vein, and the injection was carried out once in total, and the mice were sacrificed on the 42nd day.

[0208] 2. Joint swelling score

[0209] Starting from the 21st day after the primary immunization, three independent examiners performed joint swelling scores every 3 days. The severity of arthritis in each of the four paws was graded on a scale of 0 - 4: 0 points: normal; 1 point: erythema and slight swelling of the ankle joint; 2 points: erythema and slight swelling of the ankle joint or metacarpophalangeal joint; 3 points: erythema and moderate swelling of the ankle joint or metacarpophalangeal joint; 4 points: severe swelling of the ankle joint. The maximum score was 16 points.

[0210] The measurement results showed ( Figure 3 ), compared with normal mice, the joints of CIA mice were significantly swollen ( P <0.001), while the mice in the mesenchymal stem cell injection group could significantly reduce the joint swelling score of the mice in the CIA model group ( P <0.05). It is shown that the mesenchymal stem cell injection (P1 - P3) provided by the present invention has a significant therapeutic effect on the treatment of rheumatoid arthritis with a single treatment.

[0211] 3. Detection of serum inflammatory factors

[0212] On the 42nd day, blood was collected from the eyes of DBA / 1 mice, centrifuged at 2000 g for 20 min at 4°C, and the supernatant was taken. ELISA kits were used to measure the contents of IFN - γ, IL - 6, and TNF - α.

[0213] Figures 4 - 6 It was shown that the levels of IFN - γ, IL - 6, and TNF - α in the serum of the mice in the CIA model group were significantly increased compared with those of the mice in the normal control group ( P(<0.01), indicating the successful establishment of the CIA model. The mesenchymal stem cell injection (P1 - P3) can restore the levels of IFN-γ, IL-6, and TNF-α to normal levels ( P (0.01 - 0.05). Among them, the mesenchymal stem cell injection in group P1 has a better inhibitory effect on IL-6 and TNF-α.

[0214] Example 4 Evaluation of the Effect of Mesenchymal Stem Cell Injection on Mice with Lupus Erythematosus

[0215] 1. Grouping and Administration of the SLE Model

[0216] In this example, 45 female MRL / MpJ-Faslpr mice (SLE model mice) at 7 - 8 weeks old and 10 C57BL / 6 mice as normal controls (both purchased from Shanghai Slack Experimental Animal Co., Ltd.) were used. Forty mice with a urinary protein value ≥ 0.3 g / L were selected and randomly divided into 4 groups according to body weight and urinary protein content (to make the initial values of each group of animals similar): model control group, mesenchymal stem cell treatment groups (group P1, group P2, and group P3); among them, the three mesenchymal stem cell treatment groups were intravenously injected with P1 - P3 mesenchymal stem cell injections at a dose of 2x10 6 cells / mouse on the 0th, 14th, and 28th days of the experiment; the normal control group and the model control group were intravenously injected with the same volume of normal saline at the same time.

[0217] 2. SLE Clinical Observation Score

[0218] During the experiment, the skin ulceration status of the animals was observed, recorded, and evaluated weekly, and at the same time, the swelling of the main lymph nodes was observed for clinical observation scoring; the criteria are shown in Table 7 below:

[0219] Table 7 SLE Clinical Observation Score

[0220]

[0221] Figure 7 For the SLE clinical observation score, it can be seen from the results that the MRL / MpJ-Faslpr mice began to show ulceration and swelling in different parts on the 14th day of the experiment, gradually showing the clinical skin symptoms of lupus erythematosus (P < 0.01); after treatment with the mesenchymal stem cell injection (P1 - P3), the clinical score of SLE mice can be significantly reduced (P < 0.05).

[0222] 3. Detection of Anti-double-stranded DNA IgG in SLE Mice

[0223] Before administration, 2 weeks, 4 weeks, and 6 weeks (experimental endpoint) after administration, blood samples were collected from the animals in the model control group, the mesenchymal stem cell administration group, and the normal control group. A commercial ELISA kit (Mouse Anti-double-stranded DNA IgG Antibody Detection Kit, Chondrex) was used to detect the level of mouse anti-double-stranded DNA IgG antibody. Figure 8 Figure 8 shows the detection results of anti-double-stranded DNA IgG in SLE mice. It can be seen from the results that on the 28th and 42nd days of the experiment, the serum anti-double-stranded DNA IgG values of MRL / MpJ-Faslpr mice were significantly higher than those of the normal control group (P<0.001). After treatment with mesenchymal stem cell injection, the double-stranded anti-DNA IgG value was significantly reduced (P<0.01), indicating that the test drug has an inhibitory effect on the level of anti-double-stranded DNA IgG in the body caused by lupus erythematosus.

[0224] Example 5 Evaluation of the Effect of Mesenchymal Stem Cell Injection on Psoriasis Mice

[0225] In this example, C57 BL / 6 mice were used to construct a psoriasis (Ps) model by topical application of imiquimod (IMQ). After shaving the backs of all mice, 50 mg of imiquimod ointment (Mingxin Lidi, Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied topically to the back every day. The first day of application was recorded as D0, and the modeling was continued for 7 days (D6). The Ps model group was randomly divided into 4 groups: the model control group, the mesenchymal stem cell treatment groups (Group P1, Group P2, and Group P3); another group of C57 mice with shaved backs and topical application of normal saline every day was used as the normal control group. The mesenchymal stem cell treatment groups were administered subcutaneously on D1 and D4, and the administration dose was 2x10 6 / mouse. The model control group and the normal control group were given the same volume of normal saline. During the experiment, the survival and health status of the animals were observed, and the skin inflammation and related indicators were clinically scored according to the degree of skin keratinization and inflammatory cell infiltration. The animals were euthanized on D7 for relevant tests: the skin of the modeling part of the mice was collected, and the skin thickness of each group of animals was measured using a vernier caliper.

[0226] Skin scoring criteria: The skin of the animals (ears and front and hind paws) was scored. A comprehensive score was calculated based on erythema, scales, and thickness. Each index was scored into 5 grades, ranging from 0 to 5 points, where 0 points represented no relevant symptoms; 1 point represented mild symptoms; 2 points represented moderate symptoms; 3 points represented significant symptoms; 4 points represented very significant or severe symptoms, and the total score of the 3 indexes was calculated as the final score. The skin clinical score was as follows Figure 9As shown, IMQ can cause skin damage in mice, increase the degree of rash and desquamation, thicken the skin epidermis, and histopathologically show a dermis mainly composed of parakeratosis and inflammatory leukocyte infiltration; the skin clinical score of the model group increased significantly (P<0.001), while after subcutaneous injection of mesenchymal stem cell injection, the skin clinical score of mice decreased significantly (P<0.05). Skin thickness was measured at the end of the experiment, as Figure 10 shown, it was found that IMQ could significantly increase the skin thickness of model mice, while the skin thickness of mice decreased significantly after subcutaneous injection of mesenchymal stem cell injection (P<0.01), and the treatment effect of group P1 was the best.

[0227] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A mesenchymal stem cell injection, characterized in that, The preparation method of the mesenchymal stem cell injection comprises the following steps: S1. After the umbilical cord tissue is placed in a culture flask for incubation, complete culture medium - 1 is added for culture and passage to obtain P0 generation human umbilical cord mesenchymal stem cells; S2. The P0 generation human umbilical cord mesenchymal stem cells are cultured and passaged with complete culture medium - 2 to obtain P1 generation human umbilical cord mesenchymal stem cells; S3. The P1 generation human umbilical cord mesenchymal stem cells are cultured and passaged with complete culture medium - 3 to obtain P2 generation human umbilical cord mesenchymal stem cells. After centrifugation, the supernatant is discarded, and the cells are collected as seed bank cells; S4. The seed bank cells are continuously cultured to P5 generation with complete culture medium - 3 and cryopreserved to obtain mesenchymal stem cell working bank cells; S5. The mesenchymal stem cell working bank cells are resuspended with cell preservation solution to obtain mesenchymal stem cell injection; The complete culture medium - 1 described in step S1 is: a basal medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 10 - 100 μg / L SDF - 1α, and 0.35 - 0.80 mg / L folic acid; The complete culture medium - 2 described in step S2 is: a basal medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 10 - 100 μg / L SDF - 1α, 0.35 - 0.80 mg / L folic acid, and 0.5 - 2 mg / L hydrogen; The complete culture medium - 3 described in steps S3 and S4 is: a basal medium containing 10% - 15% v / v FBS, 5 - 10 μg / L bFGF, 1 - 2 mg / L methyl - β - cyclodextrin, and 0.35 - 0.80 mg / L folic acid; The culture conditions described in step S1 include 5% CO2 and 1% - 2% O2; The culture conditions described in step S2 include 5% CO2 and 5% - 10% O2; The culture conditions described in step S3 include 5% CO2 and 18% - 20% O2; The cell preservation solution described in step S5 is composed of human albumin, heparin, dextran - 40, and compound electrolyte solution.

2. The mesenchymal stem cell injection according to claim 1, wherein The cell preservation solution described in step S5 includes 10 - 20 mg / mL human albumin, 50 - 100 IU / mL heparin, and 50 - 100 mg / mL dextran - 40.

3. The mesenchymal stem cell injection according to claim 2, wherein The complete culture medium - 1 described in step S1 is: a basal medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF - 1α, and 0.35 mg / L folic acid; The complete culture medium - 2 described in step S2 is: a basal medium containing 10% v / v FBS, 7.5 μg / L bFGF, 50 μg / L SDF - 1α, 0.35 mg / L folic acid, and 1 mg / L hydrogen; The complete culture medium - 3 described in steps S3 and S4 is: a basal medium containing 10% v / v FBS, 7.5 μg / L bFGF, 1 mg / L methyl - β - cyclodextrin, and 0.35 mg / L folic acid; The cell preservation solution described in step S5 comprises 10 mg / mL human serum albumin, 50 IU / mL heparin, and 60 mg / mL dextran-40.

4. The mesenchymal stem cell injection according to claim 1, wherein The basal culture medium described above comprises DMEM / F12 culture medium, DMEM culture medium, MSC growth medium, RPMI 1640, or E8.

5. The mesenchymal stem cell injection according to claim 4, wherein The basal culture medium is DMEM / F12 culture medium.

6. The mesenchymal stem cell injection according to claim 1, characterized in that, Step S1 includes: After the umbilical cord tissue is incubated in a culture flask with 5% CO2 for 24 h, complete culture medium -1 is added and cultured and passaged under the conditions of 5% CO2 and 1%-2% O2 to obtain passage 0 human umbilical cord mesenchymal stem cells.

7. The mesenchymal stem cell injection according to claim 6, wherein Step S1 includes: After the umbilical cord tissue is incubated in a culture flask with 5% CO2 for 24 h, complete culture medium -1 is added and cultured and passaged under the conditions of 5% CO2 and 1% O2 to obtain passage 0 human umbilical cord mesenchymal stem cells.

8. The mesenchymal stem cell injection according to claim 1, wherein The culture conditions described in step S2 include 5% CO2 and 5% O2.

9. The mesenchymal stem cell injection according to claim 1, wherein The culture conditions described in step S3 include 5% CO2 and 18% O2.

10. The mesenchymal stem cell injection according to claim 1, characterized in that, The density of mesenchymal stem cells in the mesenchymal stem cell injection is 0.5×10 6 -1×10 8 cells / mL.

11. The mesenchymal stem cell injection according to claim 10, wherein The density of mesenchymal stem cells in the mesenchymal stem cell injection is 1×10 6 cells / mL.

12. The method for preparing the mesenchymal stem cell injection according to any one of claims 1-11.

13. The use of the mesenchymal stem cell injection according to any one of claims 1-11 or the mesenchymal stem cell injection prepared by the preparation method according to claim 12 in the preparation of a drug containing mesenchymal stem cells.

14. The application according to claim 13, wherein The drug described above includes drugs for preventing or treating autoimmune diseases.

15. The application according to claim 14, characterized in that, The autoimmune diseases described above include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, idiopathic thrombocytopenic purpura, or autoimmune hemolytic anemia.

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