Mesenchymal stem cell injection, preparation process and application of mesenchymal stem cell injection in treatment of autoimmune diseases

Abstract

The invention provides a mesenchymal stem cell injection, a preparation process and application of the mesenchymal stem cell injection to treatment of autoimmune diseases, and belongs to the technical field of biological medicines. The mesenchymal stem cell injection provided by the invention is prepared from a complete culture solution 1, a complete culture solution 2 and a complete culture solution 3 under specific culture conditions. The mesenchymal stem cell working bank used for preparing the mesenchymal stem cell injection is good in cell safety, has better cell proliferation ability, highly expresses CD73, CD90 and CD105, does not express CD19, CD34, CD45, CD11b and HLA-DR, and has osteogenesis and adipogenesis differentiation ability and better biological efficacy. The mesenchymal stem cell injection provided by the invention meets the pharmacopoeia standard, has high HGF and sTNFR1 contents, and can play a role in preventing and treating autoimmune diseases.

Description

Technical Field

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

[0002] Human Umbilical Cord Mesenchymal Stem Cell (hUC-MSCs) has a wide range of sources, is easy to obtain, does not damage the donor, has unique immunomodulatory function, and can inhibit the functions of a variety of 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 have a strong inhibitory effect on the immune system, and are therefore widely used in the clinical treatment of immune-related diseases, such as graft-versus-host disease and rheumatoid arthritis.

[0003] Basic fibroblast growth factor (bFGF) can promote stem cell growth, proliferation and maintain stem cell stemness; stromal cell-derived factor-1α (SDF-1α) has the function of promoting stem cell homing, chemotaxis and proliferation. Its use in the early stage of mesenchymal stem cell isolation and culture can accelerate the speed of stem cell isolation and acquisition from tissues such as the umbilical cord and help maintain stem cell activity; folic acid (vitamin B9) plays an important role in cell DNA synthesis and cell division; and 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 studies have shown 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 the body's homeostasis and the 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 development of autoimmune diseases such as rheumatoid arthritis and ulcerative colitis. In general, the inflammation, apoptosis 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 tyrosine kinase activity. After being activated by ligands, it mediates cell information transmission and is an important regulator of cell proliferation, differentiation and motility. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that can induce a variety of cell responses, including proliferation, migration and survival, by activating its receptor c-met. Studies have shown that the HGF / c-met signaling pathway has pro-inflammatory effects and can directly affect leukocyte responses in inflammation. Therefore, targeted inhibition of the HGF / c-met pathway may be a potential treatment for immune-related diseases.

[0006] At present, the research on the clinical treatment of immune-related diseases with umbilical cord mesenchymal stem cells 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 an application for treating 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 basic culture medium containing FBS, bFGF, SDF-1α and folic acid; the complete culture medium-2 includes: a basic culture medium containing FBS, bFGF, SDF-1α, folic acid and hydrogen; the complete culture medium-3 includes: a basic culture medium containing FBS, bFGF, methyl-β-cyclodextrin and folic acid. The mesenchymal stem cell working bank cells used in the preparation of mesenchymal stem cell injection of the present invention have good safety, good cell proliferation ability, high expression of CD73, CD90, CD105, no expression of CD19, CD34, CD45, CD11b, HLA-DR, and have osteogenic and adipogenic differentiation ability and 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 an effect of preventing and treating autoimmune diseases.

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

[0009] In one aspect, the present invention provides a mesenchymal stem cell injection, wherein the preparation method of the mesenchymal stem cell injection comprises the following steps:

[0010] S1, after the umbilical cord tissue is placed in a culture bottle for incubation, complete culture medium-1 is added for culture and passage to obtain P0 generation human umbilical cord mesenchymal stem cells;

[0011] S2 and P0 generation human umbilical cord mesenchymal stem cells were cultured and subcultured with complete culture medium-2 to obtain P1 generation human umbilical cord mesenchymal stem cells;

[0012] S3 and P1 generation human umbilical cord mesenchymal stem cells were cultured and subcultured with complete culture medium-3 to obtain P2 generation human umbilical cord mesenchymal stem cells. After centrifugation, the supernatant was discarded and the cells were collected as seed bank cells;

[0013] S4. The seed bank cells are cultured with complete culture medium-3 until P5 and frozen to obtain mesenchymal stem cell working bank cells;

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

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

[0016] Preferably, the complete culture medium-1 in step S1 comprises: a basic 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] Further preferably, the complete culture medium-1 in step S1 comprises: a basic 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 basic culture medium containing FBS, bFGF, SDF-1α, folic acid and hydrogen.

[0019] Preferably, the complete culture medium-2 in step S2 comprises: a basic 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] Further preferably, the complete culture medium-2 described in step S2 comprises: a basic 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 step S3 and step S4 includes: a basic culture medium containing FBS, bFGF, methyl-β-cyclodextrin and folic acid.

[0022] Preferably, the complete culture medium-3 described in step S3 and step S4 comprises: a basic 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] Further preferably, the complete culture medium-3 described in step S3 and step S4 comprises: a basic 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] Further preferably, the basal culture medium is DMEM / F12 culture medium.

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

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

[0028] Specifically, step S1 includes: placing the umbilical cord tissue in a culture bottle and incubating it at 5% CO2 for 24 hours, adding complete culture medium-1 and culturing and passage under the conditions of 5% CO2, 1%-2% O2 to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0029] Preferably, step S1 comprises: placing the umbilical cord tissue in a culture bottle and incubating it in 5% CO2 for 24 hours, adding complete culture medium-1 and culturing and passage under the conditions of 5% CO2 and 1% O2 to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0030] Specifically, the culturing in step S1 includes: adding 5 mL of complete culture medium-1, and after overnight culturing, adding 10 mL of complete culture medium-1 to continue culturing.

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

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

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

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

[0035] Specifically, the cell preservation solution in step S5 consists of human serum albumin, heparin, dextran-40 and a compound electrolyte solution.

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

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

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

[0039] Preferably, the density of mesenchymal stem cells in the mesenchymal stem cell injection 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.

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

[0042] S1, after the umbilical cord tissue is placed in a culture bottle for incubation, complete culture medium-1 is added for culture and passage to obtain P0 generation human umbilical cord mesenchymal stem cells;

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

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

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

[0046] S5. Resuspend the mesenchymal stem cell working bank cells with cell preservation solution to obtain a mesenchymal stem cell injection solution.

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

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

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

[0050] Specifically, the detection of biological efficacy 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, the mesenchymal stem cell injection prepared by the preparation method, or the mesenchymal stem cell injection that meets the standards of the cell activity identification method in the preparation of a drug containing mesenchymal stem cells.

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

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

[0056] More preferably, the autoimmune disease is rheumatoid arthritis and systemic lupus erythematosus.

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

[0058] (1) The mesenchymal stem cell working bank cells used for preparing mesenchymal stem cell injection of the present invention have good safety, good cell proliferation ability, high expression of CD73, CD90, CD105, no expression of CD19, CD34, CD45, CD11b, HLA-DR, and have osteogenic and adipogenic differentiation ability and good biological efficacy.

[0059] (2) The mesenchymal stem cell injection provided by the present invention complies with the standards of the Pharmacopoeia of the People's Republic of China, has high content of HGF and sTNFR1, and can play a role in preventing and treating autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 For cell proliferation ability.

[0061] Figure 2 For cell differentiation ability.

[0062] Figure 3 The joint swelling scores of CIA mice are shown in Table 1. In the figure, *** represents P < 0.001, and # represents P < 0.05.

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

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

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

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

[0067] Figure 8 These are the results of anti-double-stranded DNA IgG detection in SLE mice. In the figure, *** represents P<0.001, and ## represents P<0.01.

[0068] Fig. 9 is the clinical score of psoriasis mice, and # represents P<0.05.

[0069] Fig.10 The results of skin thickness of psoriasis mice. In the figure, ** represents P<0.01, and ## represents P<0.01. DETAILED DESCRIPTION

[0070] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

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

[0072] Table 1 Experimental materials

[0073] name factory Part Number Mouse IFN-γ ELISA Kit Thermo Fisher BMS606-2 Mouse IL-6 ELISA Kit Thermo Fisher KMC0061 Mouse TNF-α ELISA Kit Thermo Fisher BMS607-3 HGF ELISA Kit abcam ab275901 sTNFR1 ELISA Kit R&D DRT100

[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 comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0078] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 1% O2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0079] 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 in an incubator at 37° C. with a 5% CO 2 , 5% O 2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0080] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in an incubator with 5% CO2 and 18% O2 for 48-72 hours, which was recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0081] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze them to obtain mesenchymal stem cell working bank cells (referred to as W1). The culture system and environment are: 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 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.

[0085] 1.2 Preparation of Mesenchymal Stem Cell Working Bank Cell 2

[0086] S1. Human umbilical cord tissue comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0087] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 2% O2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion 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 in an incubator at 37°C with a 5% CO2, 10% O2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0089] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in an incubator with 5% CO2 and 20% O2 for 48-72 hours, which was recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0090] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze them to obtain mesenchymal stem cell working bank cells (referred to 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 step S3 and step 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 Cell 3

[0095] S1. Human umbilical cord tissue comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0096] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 1% O2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0097] S2. Centrifuge the P0 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 them in an incubator at 37° C. with a 5% CO 2 , 7.5% O 2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0098] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in an incubator with 5% CO2 and 18% O2 for 48-72 hours, which was recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0099] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze them to obtain mesenchymal stem cell working bank cells (referred to 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 step S3 and step 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 Cell 4

[0104] The difference between mesenchymal stem cell working bank cell 4 (W4) and mesenchymal stem cell working bank cell 1 (W1) is that 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 step S3 and step 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 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 step S3 and step 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 that the complete culture medium-1 and the 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 Cells 7

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

[0119] 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 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 comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0122] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0123] S2. Centrifuge the P0 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 in an incubator at 37° C. in a 5% CO 2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0124] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in a 5% CO2 incubator for 48-72 hours, which were recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0125] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze them to obtain mesenchymal stem cell working bank cells (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 comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0131] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 5% O2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion 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 in an incubator at 37°C with a 5% CO2, 15% O2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0133] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in an incubator with 5% CO2 and 20% O2 for 48-72 hours, which was recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0134] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze 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 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.

[0138] 1.10 Preparation of Mesenchymal Stem Cell Working Bank Cell 10

[0139] S1. Human umbilical cord tissue comes from healthy fetuses born by caesarean section in cooperative obstetrics and gynecology hospitals. 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, and do not wash it to preserve the original environment of the tissue; then directly inoculate each umbilical cord tissue piece into a T75 culture bottle, place it in a 5% CO2 incubator at a temperature of 38°C and incubate it for 24 hours. No cell culture medium is added during this process;

[0140] After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2 incubator at 38°C, 5 mL of complete culture medium-1 was added to the culture bottle, and 10 mL of complete culture medium-1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. When the cell fusion degree reached 80%, trypsin digestion solution was used for digestion to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0141] S2. Centrifuge the P0 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 in an incubator at 37° C. in a 5% CO 2 environment for subculture for 48-72 hours, which is recorded as P1 generation.

[0142] S3, the P1 generation human umbilical cord mesenchymal stem cells obtained in step S2 were centrifuged at 400g×5min, the supernatant was discarded, complete culture medium-3 was added, and the cells were subcultured at 37°C in a 5% CO2 incubator for 48-72 hours, which were recorded as P2 generation. The obtained P2 generation human umbilical cord mesenchymal stem cells were centrifuged at 400g×5min, the supernatant was discarded, and the cells were collected as a seed bank.

[0143] S4. Take the seed bank cells described in step S3, continue to culture them with complete culture medium-3 to P5 generation and freeze 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 step S3 and step 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] The mesenchymal stem cell working bank cells 1 to 10 prepared above were taken for bacterial, mycoplasma, and viral safety tests; cell morphology, cell proliferation, flow cytometry phenotype, osteogenic and adipogenic differentiation ability, and biological efficacy (HGF, sTNFR1) tests.

[0149] 2.1 Sterility, mycoplasma and virus safety testing

[0150] Sterility testing was performed according to the sterility test method in General Rules 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0151] Mycoplasma testing was performed according to the first method in General Rules 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0152] According to the kit instructions, PCR method was used to complete the detection of EBV, HCMV, HIV and other viruses in the working library cells.

[0153] The test results showed that the bacteria, mycoplasma and virus of mesenchymal stem cell working bank cells 1 to mesenchymal stem cell working bank cells 10 were all negative.

[0154] 2.2 Cell morphology and cell proliferation assay

[0155] An inverted microscope was used to observe the cell status of mesenchymal stem cell working bank cells 1 (W1) to mesenchymal stem cell working bank cells 10 (W10). The results showed that W1-W10 cells were in good condition, with single cells in a fusiform shape and fused growth in a spindle shape.

[0156] Mesenchymal stem cell working bank cells 1 (W1) to mesenchymal stem cell working bank cells 10 (W10) were taken and inoculated into a 96-well plate at 10,000 cells per well. Cell proliferation was determined by CCK-8 analysis at 0h, 24h, 48h, 72h, and 96h after inoculation. The results are shown in Figure 1As shown, W1-W3 had the strongest cell proliferation ability, W4-W9 had poor proliferation ability, and W10 had the worst proliferation ability.

[0157] 2.3 Flow cytometry phenotyping

[0158] Take cells 1 to 10 from the mesenchymal stem cell working bank and perform the following operations: collect 5×10 6 The cells were evenly divided into 10 centrifuge tubes and centrifuged at 300 g for 5 min. The supernatant was discarded and the following antibodies were added 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 Company, USA). Cell surface antigens were detected by BD Calibur flow cytometer.

[0159] The measurement results are shown in Table 2: The mesenchymal stem cell working bank cells (W1-W10) all highly expressed CD73, CD90, and CD105, and did 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 Osteogenic and adipogenic differentiation ability detection

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

[0164] Each group of cells is *10 4 / well inoculated into 224-well plates, and when the cell confluence reached about 80%, the adipogenic and osteogenic induction medium was replaced every 2-3 days. On the 21st day of induction culture, the cells were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, and then stained with 10% Oil Red-O to observe the adipogenic differentiation under a microscope, and stained with Alizarin Red solution to observe the osteogenic differentiation under a microscope. The test results are shown in Figure 2 As shown, each group of cells has the ability to differentiate into osteoblasts and adipocytes, among which W1 and W2 cells have the strongest osteoblastic and adipogenic differentiation abilities, followed by W3, W8, and W9, while W4-W7 have relatively weak differentiation abilities, and W10 has the weakest differentiation ability.

[0165] 2.5 Biological efficacy testing

[0166] The mesenchymal stem cell working bank cells (W1-W10) were taken and inoculated into 6-well plates respectively. After culturing for 48 hours, the supernatant was taken and the cells were digested and counted. The ELISA method (R&D systems product) was used to detect the content of HGF and sTNFR1 in the supernatant (Table 3).

[0167] Through detection, it was found that the HGF and sTNFR1 secreted by cells in group W1 were the highest, followed by W2 and W3, W4-W6 were lower, and W10 was the lowest.

[0168] Table 3 HGF, sTNFR1 content

[0169] Mesenchymal stem cells <![CDATA[HGF(ng / L*10 6 cell)]]> <![CDATA[sTNFR1(pg / L*10 6 cell)]]> W1 187.89 241.33 W2 180.17 236.19 W3 181.67 236.06 W4 150.66 196.08 W5 146.10 214.18 W6 148.69 206.85 W7 130.60 213.91 W8 145.28 216.41 W9 149.68 219.98 W10 110.04 191.44

[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 Solution 1

[0173] The mesenchymal stem cell working bank cells 1 prepared in Example 1 were resuscitated and cultured to 80% cell confluence, then digested with trypsin, terminated, centrifuged, washed with a compound electrolyte solution 3 times, and resuspended with a compound electrolyte solution containing 10 mg / mL human albumin, 50 IU / mL heparin and 60 mg / mL dextran-40 to prepare a mesenchymal stem cell injection 1 (P1). The injection volume was 20 mL, and the number of cells was 1E+7.

[0174] 1.2 Preparation of Mesenchymal Stem Cell Injection Solution 2

[0175] The mesenchymal stem cell working bank cells 2 prepared in Example 1 were resuscitated and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with a compound electrolyte solution for 3 times, 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 a mesenchymal stem cell injection 2 (P2).

[0176] 1.3 Preparation of Mesenchymal Stem Cell Injection Solution 3

[0177] The mesenchymal stem cell working bank cells 3 prepared in Example 1 were resuscitated and cultured to 80% cell confluence, then digested with trypsin, centrifuged, washed with a compound electrolyte solution for 3 times, 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 a mesenchymal stem cell injection 3 (P3).

[0178] 2. Detection of Mesenchymal Stem Cell Injection

[0179] 2.1 Visible foreign matter inspection

[0180] Visible foreign matter is detected according to the Visible Foreign Matter Inspection Method according to General Chapter 0904 of Part III of the 2020 Edition of the Pharmacopoeia of the People's Republic of China.

[0181] 2.2 Insoluble particle inspection

[0182] The insoluble particles were inspected according to the first method of Part III 0903 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0183] 2.3 Filling quantity inspection

[0184] Carry out filling quantity inspection according to General Rule 0102 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0185] 2.4 Osmotic pressure test

[0186] The osmotic pressure molar concentration was determined according to General Rule 0632 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0187] 2.5 Cell number and cell viability

[0188] A cell counter was used to count cells, and 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 testing

[0190] Sterility testing was performed according to the sterility test method in General Rules 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0191] 2.7 Mycoplasma Detection

[0192] Mycoplasma testing was performed according to the first method in General Rules 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0193] 2.8 Endotoxin detection

[0194] Endotoxin testing was performed according to the gel method in General Chapter 1143 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0195] 2.9 HGF and sTNFR1 results

[0196] The mesenchymal stem cell injection solution (P1-3) obtained in Example 2 was divided into 1*10 4 / cm 2 Inoculate T75 culture flasks, culture for 48 hours, harvest the supernatant, digest and count the cells, and detect the HGF and sTNFR1 content in the samples according to the instructions of the HGF and sTNFR1 kits.

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

[0198] Table 4 Pharmacopoeia standards

[0199]

[0200] Table 5 Test results of mesenchymal stem cell injection

[0201]

[0202]

[0203] Table 6 Results of mesenchymal stem cell injection HGF and sTNFR1

[0204] Mesenchymal stem cell injection <![CDATA[HGF(ng / *10 6 cell)]]> <![CDATA[sTNFR1(pg / *10 6 cell)]]> P1 190.35 251.62 P2 176.86 243.11 P3 188.43 238.75

[0205] Example 3 Evaluation of the effect of mesenchymal stem cell injection on CIA mice

[0206] 1. CIA model construction and drug administration

[0207] This example uses 50 6-8 week old male DBA / 1 mice for the experiment. The DBA / 1 mice were randomly divided into 5 groups, namely a normal control group, a model group, and a mesenchymal stem cell injection group (P1-P3), with 10 mice in each group. The model group mice were modeled as follows: an equal volume of a bovine type II collagen solution with a concentration of 4 mg / mL and complete Freund's adjuvant were mixed, and the injection emulsion was obtained after sufficient emulsification for intradermal injection at the base of the tail. The model group and the mesenchymal stem cell injection group were intradermally injected with 100 μL of injection emulsion at the base of the mouse tail on the 1st day and the 21st day of the experiment, respectively. The normal control group was intradermally injected with 100 μL of PBS solution at the base of the mouse tail on the 1st day and the 21st day of the experiment.

[0208] On the 22nd day of the experiment, the mesenchymal stem cell treatment group was given 2x10 6 The mice were injected with mesenchymal stem cell solution intravenously (P1-P3), and the normal control group and model group were injected with an equal volume of saline through the tail vein. The mice were injected once in total and then killed on the 42nd day.

[0209] 2. Joint swelling score

[0210] Starting from the 21st day after the first immunization, three independent examiners scored joint swelling 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 palmar joint; 3 points: erythema and moderate swelling of the ankle joint or palmar joint; 4 points: severe swelling of the ankle joint. The maximum score is 16 points.

[0211] The results showed that ( Figure 3 ), compared with normal mice, CIA mice had significantly swollen joints (P<0.001), while the mice in the mesenchymal stem cell injection group were able to significantly reduce the joint swelling scores of the CIA model group mice (P<0.05). This indicates 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.

[0212] 3. Serum inflammatory factor detection

[0213] On day 42, blood was collected from the eyeballs of DBA / 1 mice and centrifuged at 2000 g for 20 min at 4°C to collect the supernatant. ELISA kits were used to measure the levels of IFN-γ, IL-6, and TNF-α.

[0214] Figure 4-Figure 6 The results showed that the levels of IFN-γ, IL-6, and TNF-α in the serum of mice in the CIA model group were significantly increased compared with those in the normal control group (P<0.01), indicating that the CIA model was successfully established. Mesenchymal stem cell injection (P1-P3) was able to 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 the P1 group had a better inhibitory effect on IL-6 and TNF-α.

[0215] Example 4 Evaluation of the effect of mesenchymal stem cell injection on lupus erythematosus mice

[0216] 1. SLE model grouping and drug administration

[0217] In this example, 45 7-8 week old female MRL / MpJ-Faslpr mice (SLE model mice) and 10 C57BL / 6 mice as normal controls (all purchased from Shanghai Slake Experimental Animal Co., Ltd.) were used. 40 mice with urine protein values ​​≥ 0.3 g / L were selected and randomly divided into 4 groups according to body weight and urine protein content (so that the initial values ​​of each group of animals were similar): model control group, mesenchymal stem cell treatment group (P1 group, P2 group and P3 group); among them, the three mesenchymal stem cell treatment groups were treated with 2×10 6The mice were intravenously injected with P1-P3 mesenchymal stem cell injection at a dose of 1:1 cell / mouse; the normal control group and the model control group were intravenously injected with the same volume of normal saline at the same time.

[0218] 2. SLE clinical observation score

[0219] During the experiment, the skin ulceration of the animals was observed, recorded and evaluated weekly, and the swelling of the main lymph nodes was observed and clinical observation scores were performed; the standards are shown in Table 7 below:

[0220] Table 7 SLE clinical observation scores

[0221]

[0222]

[0223] Figure 7 is the clinical observation score of SLE. The results show that MRL / MpJ-Faslpr mice began to develop ulcers and swelling in different parts of the body from the 14th day of the experiment, and gradually showed clinical skin symptoms of lupus erythematosus (P<0.01). Treatment with mesenchymal stem cell injection (P1-P3) can significantly reduce the clinical scores of SLE mice (P<0.05).

[0224] 3. Detection of anti-double-stranded DNA IgG in SLE mice

[0225] Blood samples were collected from the model control group, mesenchymal stem cell administration group and normal control group before administration and 2 weeks, 4 weeks and 6 weeks after administration (experimental endpoint), and the mouse anti-double-stranded DNA IgG antibody level was detected using a commercial ELISA kit (mouse anti-double-stranded DNA IgG antibody detection kit, Chondrex). Figure 8 The results of the anti-double-stranded DNA IgG test in SLE mice show that the serum anti-double-stranded DNA IgG values ​​of MRL / MpJ-Faslpr mice on the 28th and 42nd days of the experiment were significantly higher than those of the normal control group (P<0.001), and the double-stranded anti-DNA IgG values ​​were significantly reduced after treatment with mesenchymal stem cell injection (P<0.01), indicating that the test drug has an inhibitory effect on the anti-double-stranded DNA IgG level in the body caused by lupus erythematosus.

[0226] Example 5 Evaluation of the effect of mesenchymal stem cell injection on psoriasis mice

[0227] In this example, C57 BL / 6 mice were used to construct a psoriasis (Ps) model by applying imiquimod (IMQ). After shaving the back of all mice, 50 mg of imiquimod ointment (Mingxinlidi, Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back every day. The first day of application was recorded as D0, and the model was established for 7 days (D6); the Ps model group was randomly divided into 4 groups: model control group, mesenchymal stem cell treatment group (P1 group, P2 group and P3 group); C57 mice with shaved back and saline applied to the back every day were set as normal control group. The mesenchymal stem cell treatment group was treated subcutaneously on D1 and D4, with a dose of 2x10 6 / mouse, the model control group and the normal control group were given the same volume of saline. During the experiment, the survival and health 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. On D7, the animals were euthanized and related tests were performed: the skin of the modeled mice was collected, and the skin thickness of each group of animals was measured using a vernier caliper.

[0228] Skin scoring criteria: Animal skin (ears and front and back paws) is scored, and a comprehensive score is given based on erythema, scaling, and thickness. Each indicator is scored on a 5-point scale, from 0 to 5 points, where 0 means no relevant symptoms; 1 means mild symptoms; 2 means general symptoms; 3 means significant symptoms; 4 means very significant or severe symptoms, and the total score of the three indicators is calculated as the final score. Fig. 9 As shown in the results, IMQ can cause skin damage in mice, with increased rash and desquamation, thickening of the epidermis, and histopathological findings of the dermis with parakeratosis and inflammatory leukocyte infiltration. The clinical score of the skin in the model group was significantly increased (P<0.001), while the clinical score of the skin in mice was significantly reduced after subcutaneous injection of mesenchymal stem cell injection (P<0.05). Skin thickness was measured at the end of the experiment, such as Fig.10 As shown in the results, it was found that IMQ could significantly increase the skin thickness of model mice, while the skin thickness of mice was significantly reduced after subcutaneous injection of mesenchymal stem cell injection (P<0.01), among which the P1 group had the best therapeutic effect.

[0229] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

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 bottle for incubation, complete culture medium-1 is added for culture and passage to obtain P0 generation human umbilical cord mesenchymal stem cells; S2 and P0 generation human umbilical cord mesenchymal stem cells were cultured and subcultured with complete culture medium-2 to obtain P1 generation human umbilical cord mesenchymal stem cells; S3 and P1 generation human umbilical cord mesenchymal stem cells were cultured and subcultured with complete culture medium-3 to obtain P2 generation human umbilical cord mesenchymal stem cells. After centrifugation, the supernatant was discarded and the cells were collected as seed bank cells; S4. The seed bank cells are cultured with complete culture medium-3 until P5 and frozen to obtain mesenchymal stem cell working bank cells; S5, resuspending the mesenchymal stem cell working bank cells with a cell preservation solution to obtain a mesenchymal stem cell injection solution; The complete culture medium-1 described in step S1 comprises: a basic culture medium containing FBS, bFGF, SDF-1α and folic acid; The complete culture medium-2 described in step S2 comprises: a basic culture medium containing FBS, bFGF, SDF-1α, folic acid and hydrogen; The complete culture medium-3 described in step S3 and step S4 comprises: a basic culture medium containing FBS, bFGF, methyl-β-cyclodextrin and folic acid; The culture conditions in step S1 include 5% CO2, 1%-2% O2; The culture conditions in step S2 include 5% CO2, 5%-10% O2; The culture conditions in step S3 include 5% CO2, 18%-20% O2; The cell preservation solution described in step S5 is composed of human serum albumin, heparin, dextran-40 and a compound electrolyte solution.

2. The mesenchymal stem cell injection according to claim 1, characterized in that: The complete culture medium-1 described in step S1 comprises: a basic 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; The complete culture medium-2 described in step S2 comprises: a basic 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; The complete culture medium-3 described in step S3 and step S4 comprises: a basic 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; 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, characterized in that: The complete culture medium-1 described in step S1 comprises: a basic culture 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 comprises: a basic 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; The complete culture medium-3 described in step S3 and step S4 comprises: a basic 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; The cell preservation solution described in step S5 includes 10 mg / mL human albumin, 50 IU / mL heparin and 60 mg / mL dextran-40.

4. The mesenchymal stem cell injection according to claim 1, characterized in that: The basic culture medium includes DMEM / F12 culture medium, DMEM culture medium, MSC growth medium, RPMI 1640 or E8.

5. The mesenchymal stem cell injection according to claim 4, characterized in that: The basic culture medium is DMEM / F12 culture medium.

6. The mesenchymal stem cell injection according to claim 1, characterized in that: The step S1 comprises: placing the umbilical cord tissue in a culture bottle and incubating it in 5% CO2 for 24 hours, adding complete culture medium-1 and culturing and passage under the conditions of 5% CO2 and 1%-2% O2 to obtain P0 generation human umbilical cord mesenchymal stem cells.

7. The mesenchymal stem cell injection according to claim 7, characterized in that: The step S1 comprises: placing the umbilical cord tissue in a culture bottle and incubating it in 5% CO2 for 24 hours, adding complete culture medium-1 and culturing and subculturing under the conditions of 5% CO2 and 1% O2 to obtain P0 generation human umbilical cord mesenchymal stem cells.

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

9. The mesenchymal stem cell injection according to claim 1, characterized in that: The culture conditions described in step S3 include 5% CO2, 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, characterized in that: The density of mesenchymal stem cells in the mesenchymal stem cell injection is 1×10 6 cells / mL.

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

13. The method for identifying cell activity of a mesenchymal stem cell injection according to any one of claims 1 to 11, characterized in that: The cell activity identification method comprises detecting the cell morphology, cell viability, flow cytometry phenotype, osteogenic and adipogenic differentiation ability or biological efficacy of mesenchymal stem cells.

14. The method for identifying cell activity according to claim 13, characterized in that: The flow cytometry phenotype detection includes detecting one or more of CD73, CD90, CD105, CD19, CD34, CD45, CD11b, and HLA-DR.

15. The method for identifying cell activity according to claim 13, characterized in that: The detection of biological efficacy includes detection of HGF or sTNFR1.

16. Use of the mesenchymal stem cell injection according to any one of claims 1 to 11, the mesenchymal stem cell injection prepared by the preparation method according to claim 12, or the mesenchymal stem cell injection that meets the standards of the cell activity identification method according to any one of claims 13 to 15 in the preparation of a drug containing mesenchymal stem cells.

17. The use according to claim 16, characterized in that The medicine includes medicine for preventing or treating autoimmune diseases.

18. The use according to claim 17, characterized in that The autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, idiopathic thrombocytopenia, or autoimmune hemolytic anemia.

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