Pretreatment method for improving immunomodulatory function of umbilical cord mesenchymal stem cells and application

Abstract

The invention discloses a pretreatment method for improving the immunomodulatory function of umbilical cord mesenchymal stem cells. The pretreatment method comprises the steps of cell separation, cell culture and cell activation. The immunoregulation capability of the umbilical cord mesenchymal stem cells is improved through creatine pretreatment and IFN-gamma cell factor activation. According to the pretreatment method, the mRNA expression level of COX2 and IDO-1 in the umbilical cord mesenchymal stem cells can be improved, the proportion of T cell subsets in PBMC can be regulated and controlled, and the proportion of CD4 and CD8 native T cells and Tregs cells in the spleen can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells and an application thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by T and B cell overactivation, autoantibody production, immune complex deposition and multi-organ damage, and has genetic susceptibility. Currently, allogeneic mesenchymal stromal cells (MSCs) derived from bone marrow or umbilical cord can be used to treat refractory systemic lupus erythematosus (SLE) patients. After long-term follow-up, patients tolerated MSCs well, and no transplant-related adverse events occurred. During the 12-month follow-up period, the SLE disease activity index (SLEDAI) score and British Isles lupus assessment group index (BILAG) of patients were reduced, urine protein decreased, and renal function was improved. The one-year complete remission rate of patients was 32.5%, and the overall clinical remission rate could reach 60%. However, in the long run, the overall and partial clinical remission rate of 5 years was only 34%, and 24% of patients still relapsed after remission. Therefore, although MSC therapy has gradually developed into one of the ideal treatment options for refractory lupus nephritis, its effectiveness needs to be improved urgently. Summary of the invention

[0003] In order to solve the existing technical problems, the present invention provides a pretreatment method and application for increasing the immunomodulatory function of umbilical cord mesenchymal stem cells. The specific scheme is as follows: In one aspect, the present invention provides a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells, characterized in that it comprises the following steps: S1. Cell separation: Take 5-10 cm of the umbilical cord of the newborn, cut it and immediately place it in sterile DMEM / F12 culture medium; use PBS to rinse off the residual blood and clots on the surface of the umbilical cord, cut the umbilical cord into small pieces of 2-3 cm in a clean bench, remove the artery and vein on the umbilical cord, wash it again with PBS, and peel off Wharton's jelly; S2. Cell culture: Cut Wharton's jelly into 2 mm pieces 3Small pieces of MSC were spread on the bottom of the culture dish, and DMEM / F12 medium containing 10% fetal bovine serum by mass was added to the medium. Cell culture was carried out at 37°C, and fresh medium was replaced every 72-96 hours. After 14-21 days, primary cells were observed to grow around the tissue under a microscope and form colonies. They were digested with trypsin solution for 2 minutes and subcultured. When the cell fusion reached 80%, subculture was carried out again until the 6th generation of MSC was cultured. S3. Cell activation: When the cell confluence is 80-90%, use DMEM / F12 medium without fetal bovine serum to culture the cells, add 1% by volume of penicillin-streptomycin for screening, add creatine for pretreatment, and use IFN-γ cytokine to activate for 12 hours to obtain activated umbilical cord mesenchymal stem cells.

[0004] Preferably, the pancreatic enzyme solution in step S2 is a pancreatic enzyme solution with a mass percentage of 0.25%, including 0.01 mass% EDTA.

[0005] Preferably, the creatine pretreatment and IFN-γ cytokine activation treatment in step S3 can produce an additive effect.

[0006] On the other hand, the present invention provides an application of a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells, wherein the mesenchymal stem cells obtained by the pretreatment method are used to prepare a mesenchymal stem cell preparation.

[0007] Preferably, the mesenchymal stem cells obtained by the pretreatment method are used to prepare drugs.

[0008] Preferably, the mesenchymal stem cells prepared by the pretreatment method are used to treat systemic lupus erythematosus.

[0009] Preferably, the IFN-γ cytokine in the pretreatment method can respectively increase the mRNA expression levels of COX2 and IDO-1 in umbilical cord mesenchymal stem cells, and the co-treatment of creatine combined with IFN-γ cytokine can further increase the mRNA expression levels of COX2, IL-10 and IDO in umbilical cord mesenchymal stem cells on the basis of the level of IFN-γ activation.

[0010] Preferably, the pretreatment method can enhance the proliferation inhibitory function of umbilical cord mesenchymal stem cells on CD4 and CD8 T cells.

[0011] Preferably, the pretreatment method is capable of regulating the proportion of T cell subsets in PBMC.

[0012] Preferably, the pretreatment method can improve the phenotype of the lupus model and increase the proportion of CD4, CD8 naive T cells and Tregs cells in the spleen in the lupus mouse model.

[0013] Beneficial effects: 1. Enhance the expression of immunomodulatory molecules: IFN-γ cytokine can significantly increase the mRNA expression levels of COX2 and IDO-1 in umbilical cord mesenchymal stem cells, while co-treatment with creatine combined with IFN-γ cytokine can further increase the mRNA expression levels of COX2, IL-10 and IDO on this basis. These effector molecules play a key role in the immune regulation process, such as participating in the inhibition of NK cell proliferation, regulating Th1, Th17 and Treg cells, and regulating B cell activation and proliferation, thereby strengthening the core mechanism of the immune regulation function of umbilical cord mesenchymal stem cells.

[0014] 2. Effective inhibition of T cell proliferation: After pretreatment with 1000μM creatine and 20ng / ml IFN-γ, the proliferation inhibition function of umbilical cord mesenchymal stem cells on CD4 and CD8 T cells was significantly enhanced. In related experiments, by co-culturing the patient's mononuclear cells with the pretreated umbilical cord mesenchymal stem cells, it can be clearly observed that T cell proliferation is effectively inhibited, which is of great significance for the regulation of T cell overactivation in autoimmune diseases and helps to restore immune balance.

[0015] 3. Precise regulation of the proportion of T cell subsets: The pretreatment method of the present invention has significant results in regulating the proportion of T cell subsets in PBMC. Compared with the MSC + IFN-γ treatment group and the Cr pre-shSLC6A8-MSC + IFN-γ group, the Cr pre-MSC + IFN-γ group can significantly increase the proportion of CD4 naive T cells and reduce the proportion of CD4 Teff cells. The proportion of Treg in PBMC in this group is more significant than that in the MSC + IFN-γ group, achieving precise optimization of T cell subsets and further regulating the direction and intensity of immune response.

[0016] 4. Improvement of lupus model symptoms: In the lupus mouse model experiment, umbilical cord mesenchymal stem cells pretreated with creatine showed good therapeutic effects. The treated cells can significantly reduce the spleen and body mass index of lupus mice, significantly reduce serum autoantibody levels, reduce kidney urine protein levels and improve pathological conditions. At the same time, the proportion of CD4, CD8 naive T cells and Tregs cells in the spleen is significantly increased, providing new effective ways and methods for the treatment of autoimmune diseases such as systemic lupus erythematosus, and is expected to improve the clinical treatment effect of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figures 2 and 3 show the effects of the pretreatment method of the present invention on the characteristics of mesenchymal stem cells: A is a graph showing the results of the CCK8 cell viability test in Example 2; B is a graph showing the changes in the intracellular ATP content after creatine acts on mesenchymal stem cells in Example 3; C is a graph showing the mRNA expression level of MSC effector molecules in Example 4; D is a graph showing the identification of MSC surface markers in Example 5; E is a graph showing the detection of adipogenic differentiation ability in Example 6; and F is a graph showing the identification of the osteogenic differentiation ability of MSC pretreated with creatine and ordinary MSC in Example 6.

[0018] Figure 2 The results of the effect of the pretreatment method of the present invention on the immune regulatory function of MSC: A is a graph showing the proliferation of CD4 T cells and CD8 T cells in the untreated group, Anti-CD3 / CD28 treated group, Anti-CD3 / CD28+MSC+IFN-γ group, Anti-CD3 / CD28+MSC+Cr+IFN-γ group, and Anti-CD3 / CD28+shSLC6A8-MSC+Cr+IFN-γ group of Example 7; B is a graph showing the proportion of T lymphocyte subsets in the PBMC group, MSC+IFN-γ treated group, Cr pre-MSC+IFN-γ group, Cr pre-shSLC6A8-MSC+IFN-γ group, and Cr group of Example 8.

[0019] Figure 3 The figures are the improvement results of lupus phenotype in R848-induced lupus mice after pretreatment of umbilical cord mesenchymal stem cells with creatine in Example 9; A is the change of IFN-γ level in serum of Balb / c mice after R848 treatment; B is the spleen size and spleen weight ratio of each group of mice after MSC treatment; C is the serum anti-dsDNA antibody level of each group of mice after MSC treatment; D is the urine protein level of each group of mice after MSC treatment; E is the renal pathological changes of each group of mice after MSC treatment; F is the proportion of spleen T lymphocyte subsets of each group of mice after MSC treatment; G is the proportion of spleen Treg of each group of mice after MSC treatment. DETAILED DESCRIPTION

[0020] The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

[0021] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention.

[0022] Example 1: Method for treating umbilical cord mesenchymal stem cells This example provides a conventional method for processing umbilical cord mesenchymal stem cells.

[0023] Take 5-10cm of the umbilical cord of a healthy full-term newborn, cut it and immediately place it in a sterile DMEM / F12 (Nanjing Shenghang Biotechnology Co., Ltd., catalog number: BC-M-002) culture medium, and transfer it on ice; use sterile PBS to rinse off the residual blood and clots on the surface of the umbilical cord, cut the umbilical cord into small pieces of 2-3cm in a clean bench, cut the umbilical cord along the longitudinal axis, remove the artery and vein on the umbilical cord, wash it again with PBS, and peel off Wharton's jelly; cut the Wharton's jelly into small pieces of about 2mm3, spread it on the bottom of a 10cm culture dish, add DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, and place it in an incubator at 37℃ and 5% CO2 to stand, and replace the fresh culture medium after 72-96h; about 14-21 After one day, primary cells were observed to grow around the tissue under a microscope to form colonies. They were digested for 2 min using 0.25% (V / V) trypsin (Gibco, catalog number: 25200056) containing 0.01% EDTA, and subcultured. When the cell fusion reached 80%, they were subcultured again until the sixth generation of MSCs was cultured, at which point the preparation was complete.

[0024] The MSCs used in Examples 2-9 were all prepared based on the method of this example.

[0025] Example 2: Detection of the effect of adding different concentrations of creatine on the growth activity of umbilical cord mesenchymal stem cells In this example, the activity of umbilical cord mesenchymal stem cells was detected by changing the added creatine concentration and incubation time.

[0026] In order to detect whether different concentrations of creatine would affect the growth activity of MSC cells, the present invention inoculated 1×10 4MSCs with a certain number of cells were placed in an incubator for 24 hours; creatine reagents with concentrations of 10μM, 100μM, and 1000μM were added to the cells, and the culture plates were shaken crosswise to mix; the culture plates were incubated in the incubator for 12h and 24h, respectively; 10μL of CCK-8 (Beyotime, Product No.: C0038) solution was added to each well; the culture plates were incubated in the incubator for 2h; the absorbance value at 450nm was measured with an enzyme-labeled instrument.

[0027] The results are as follows Figure 1 As shown in A, three concentrations of creatine did not have a significant effect on the activity of umbilical cord mesenchymal stem cells at different time points.

[0028] Example 3: Detection of changes in intracellular ATP content after creatine action Since creatine can be phosphorylated to form phosphocreatine in the presence of creatine kinase, resulting in the generation of a large amount of ATP, this embodiment can reflect whether creatine is utilized by mesenchymal stem cells after addition by detecting changes in the content of intracellular ATP.

[0029] Step 1: When the umbilical cord mesenchymal stem cells reached 80-90% confluence in a 6-well plate, the cell culture medium was replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin was added, and 1000 μM creatine was added to the cell culture medium for pretreatment for 12 h; Step 2: Discard the culture medium and add lysis solution at a ratio of 200 μl per well of the 6-well plate. Use a pipette to repeatedly blow or shake the culture plate to allow the lysis solution (Beyotime, Catalog No.: S0026) to fully contact and lyse the cells. After lysis, centrifuge at 4°C 12000g for 5 minutes, take the supernatant, and then use the ATP detection kit (Beyotime, Catalog No.: S0026) to detect ATP concentration.

[0030] The results are as follows Figure 1 As shown in B, when mesenchymal stem cells were stimulated with creatine, the intracellular ATP content increased significantly.

[0031] Example 4: Selection of the optimal concentration of creatine to induce umbilical cord mesenchymal stem cells In this example, the optimal concentration of creatine to induce umbilical cord mesenchymal stem cells was selected by detecting the effects of different concentrations of creatine on the expression of effector molecules of umbilical cord mesenchymal stem cells with or without IFN-γ cytokine stimulation.

[0032] When the umbilical cord mesenchymal stem cells have a confluence of 80-90% in the culture dish, the cell culture medium is replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin is added, and 10 μM, 100 μM, and 1000 μM creatine are added for pretreatment for 12 hours, followed by activation with 20 ng / ml IFN-γ cytokine for 12 hours. In the immunomodulatory effect of MSC, effector molecules such as COX2, IL-10, and IDO are involved in the inhibition of NK cell proliferation, Th1, Th17, and Treg regulation, and the regulation of B cell activation and proliferation. Therefore, this embodiment detects the expression levels of COX2, IL-10, and IDO-1.

[0033] The results are as follows Figure 1 As shown in Figure C, IFN-γ stimulation can significantly increase the mRNA expression levels of COX2 and IDO-1 in umbilical cord mesenchymal stem cells, and has no significant effect on the expression level of IL-10. Compared with creatine concentrations of 10μM and 100μM, 1000μM creatine stimulation and IFN-γ stimulation can significantly increase the expression levels of COX2, IL-10, and IDO-1, and 1000μM creatine stimulation and IFN-γ stimulation have an additive effect.

[0034] Example 5: Effect of creatine pretreatment on identification of umbilical cord mesenchymal stem cells This example provides the identification of umbilical cord mesenchymal stem cells after creatine pretreatment.

[0035] Step 1: When the umbilical cord mesenchymal stem cell confluence is 80-90%, the cell culture medium is replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin is added, 1000 μM creatine is added for pretreatment for 12 hours, and then activated with 20 ng / ml IFN-γ cytokine for 12 hours.

[0036] Step 2: Use 0.25% trypsin (containing 0.01% EDTA) to digest the cells and add PBS to wash them.

[0037] Step 3: Incubate umbilical cord mesenchymal stem cell surface marker flow cytometry antibodies (CD11b, HLA-DR, CD19, CD73, CD90, CD105) at 4°C in the dark for 30 minutes.

[0038] Step 4: Add 200 μl PBS (Gibco, catalog number: 10010023) to the incubated cells, centrifuge at 1800 rpm, 4°C for 5 minutes, discard the liquid and resuspend with 200 μl PBS, and detect using BD LSRFortessa cell analyzer.

[0039] The results are as follows Figure 1As shown in D, creatine pretreated mesenchymal stem cells expressed positive markers CD73, CD90, and CD105 on their surface, but did not express negative markers CD11b, HLA-DR, and CD19. Figure 1 The surface marker expression pattern of untreated umbilical cord mesenchymal stem cells in C is consistent, indicating that mesenchymal stem cells pretreated with creatine can still be characterized by surface markers.

[0040] Example 6: Effect of creatine pretreatment on the differentiation function of umbilical cord mesenchymal stem cells This example identifies the differentiation function of creatine pretreatment on umbilical cord mesenchymal stem cells by inducing differentiation medium.

[0041] Step 1: When the umbilical cord mesenchymal stem cell confluence is 80-90%, the cell culture medium is replaced with serum-free DMEM / F12 medium, 1% penicillin-streptomycin is added, 1000 μM creatine (Cr) is added for pretreatment for 12 hours, and then activated with 20 ng / ml IFN-γ cytokine for 12 hours.

[0042] Step 2: The sixth generation MSCs were cultured at 5×10 5 / well inoculated into 6-well plates until the cells are 100% confluent, and commercial human umbilical cord mesenchymal stem cell adipogenic differentiation medium (Saiye Biotechnology Co., Ltd., catalog number: HUXUC-90031) is used. The medium is replaced with adipogenic induction medium A solution, maintained for 3 days and then replaced with B solution, and then replaced with A solution again after 24 hours. After 5 cycles of alternating cycles, the medium is maintained with B solution for 7 days (the medium is changed once every 2 to 3 days). The cells are fixed with 4% (V / V) paraformaldehyde and stained with Oil Red O working solution for 30 minutes. After staining, the above differentiation effect is identified under a microscope; Step 3: The sixth generation MSCs were cultured at 5×10 5 / well was inoculated in a 6-well plate pre-coated with 0.1% (M / V) gelatin. When the cells were 60%-70% confluent, the culture medium was replaced with adult umbilical cord mesenchymal stem cell osteogenic differentiation medium (Saiye Biotechnology Co., Ltd., Cat. No.: HUXUC-90021), and the medium was changed every 2-3 days. After 21 days of induction, the cells were fixed with 4% paraformaldehyde and then stained with Alizarin Red for 5 minutes.

[0043] The results are as follows Figure 1 E- Figure 1 As shown in F, Figure 1 E shows that creatine pretreatment has no effect on the adipogenic differentiation ability of umbilical cord mesenchymal stem cells. Figure 1 F shows that creatine pretreatment has no effect on the osteogenic differentiation ability of umbilical cord mesenchymal stem cells.

[0044] Example 7: Inhibitory effect of umbilical cord mesenchymal stem cells on T cell proliferation In this example, the proliferation capacity of T cells was detected by co-culturing the patient's mononuclear cells with umbilical cord mesenchymal stem cells pretreated with creatine and IFN-γ.

[0045] Step 1: When the umbilical cord mesenchymal stem cell confluence is 80-90%, the cell culture medium is replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin is added, 1000 μM creatine is added for pretreatment for 12 hours, and then activated with 20 ng / ml IFN-γ cytokine for 12 hours.

[0046] Step 2: Mononuclear cells (PBMCs) labeled with appropriate concentrations of CFSE (eBioscience, catalog number: 65-0850-84) were inoculated into MSCs stimulated by different treatments at a ratio of MSC:PBMC=1:10 for co-culture. At this time, the culture medium was replaced with RPMI 1640 culture medium containing only 2% serum, and Anti-CD3 / CD28 was added at a ratio of 1μg / ml. The cells were divided into untreated group (PBMC only), Anti-CD3 / CD28 treated group (PBMC only), Anti-CD3 / CD28+MSC+IFN-γ group (PBMC co-cultured with MSC), Anti-CD3 / CD28+MSC+Cr+IFN-γ group (PBMC co-cultured with MSC), Anti-CD3 / CD28+shSLC6A8-MSC+Cr+IFN-γ (PBMC co-cultured with MSC with knockdown of creatine transporter) group. After 48h of co-culture, PBMCs were aspirated, centrifuged and supernatant discarded, and fresh PBS was used. After rinsing once, centrifuge again and discard the supernatant, add 50μl PBS to resuspend the cells, then add dead and live dye (Thermo Fisher Scientific, Catalog No.: 65-0866-14), CD4 (BD, Catalog No.: 551980), CD8 (BD, Catalog No.: 560662) flow cytometry antibodies for staining in the dark for 30 minutes. Then add 200μl PBS to rinse once, centrifuge and discard the supernatant, resuspend the cells with 200μl PBS, and use BD LSRFortessa cell analyzer for detection.

[0047] The results are as follows Figure 2As shown in A-, when 1000 μM creatine and 20 ng / ml IFN-γ were used for pretreatment at the same time, the proliferation inhibitory function of umbilical cord mesenchymal stem cells on CD4 and CD8 T cells was significantly increased, and when the creatine transporter SLC6A8 on the umbilical cord mesenchymal stem cells was knocked down so that creatine could not act on the mesenchymal stem cells, the umbilical cord mesenchymal stem cells were treated with creatine and IFN-γ again, and their inhibitory function on T cells was reduced.

[0048] Example 8: Detection of the regulatory effect of umbilical cord mesenchymal stem cells on T cell subsets In this example, the patient's mononuclear cells were co-cultured with umbilical cord mesenchymal stem cells pretreated with creatine and IFN-γ to detect the results of umbilical cord mesenchymal stem cells regulating T cell subsets.

[0049] Step 1: When the umbilical cord mesenchymal stem cell confluence is 80-90%, the cell culture medium is replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin is added, 1000 μ m creatine is added for pretreatment for 12 h, and then activated with 20 ng / ml IFN-γ cytokine for 12 h.

[0050] Step 2: PBMCs were inoculated into MSCs stimulated by different treatments at a ratio of MSC:PBMC=1:10 for co-culture. At this time, the culture medium was replaced with RPMI 1640 culture medium containing only 2% serum, and Anti CD3 / CD28 was added at a ratio of 1μg / ml and IL-2 was added at a ratio of 5ng / ml. The cells were divided into PBMC group (PBMC only), MSC+IFN-γ treatment group (PBMC and MSC co-culture), Cr pre-MSC+IFN-γ group (PBMC and MSC co-culture), Cr pre-shSLC6A8-MSC+IFN-γ group (PBMC and MSC co-cultured with creatine transporter knockdown), and Cr group. After 48h of co-culture, the PBMCs were aspirated, centrifuged and the supernatant was discarded. The cells were rinsed with fresh PBS and centrifuged again to discard the supernatant. 50μl PBS was added. Resuspend the cells, then add live / dead dye (Thermo Fisher Scientific, Catalog No.: 65-0866-14), CD45RA (BD, Catalog No.: 555488), CD4 (BD, Catalog No.: 561839), CD4 (BD, Catalog No.: 555346), CD8 (BD, Catalog No.: 560662), CCR7 (BD, Catalog No.: 567314), CD25 (BD, Catalog No.: 567317), Foxp3 (Biolegend, Catalog No.: 320124) flow cytometry antibodies for staining in the dark for 30 minutes. Then add 200μl PBS to rinse once, centrifuge and discard the supernatant, resuspend the cells with 200μl PBS, and detect the proportion of T cell subsets in PBMC on a flow cytometer.

[0051] The results are as follows Figure 2 As shown in Figure B, compared with the MSC+IFN-γ treatment group and the Cr pre-shSLC6A8-MSC+IFN-γ group, the proportion of CD4 naive T cells in the Cr pre-MSC+IFN-γ group was significantly increased, while the proportion of CD4 Teff cells was significantly reduced or showed a significant downward trend, and the regulatory effect of the Cr pre-shSLC6A8-MSC+IFN-γ group on T cell subsets was similar to that of the MSC+IFN-γ treatment group. And the proportion of Treg in PBMC in the Cr pre-MSC+IFN-γ group was more significant than that in the MSC+IFN-γ group. In order to verify whether direct contact between creatine and T lymphocytes will cause changes in cell subsets, the present invention also set up a group of Cr groups that directly used creatine to treat PBMCs. The results showed that the Cr group had no regulatory effect on T lymphocyte subsets in PBMCs.

[0052] Example 9: Improvement of lupus phenotype in R848-induced Balb / c mice after creatine pretreatment of umbilical cord mesenchymal stem cells This example provides the results of improvement of lupus phenotype in R848-induced Balb / c mice by umbilical cord mesenchymal stem cells pretreated with creatine.

[0053] Step 1: When the umbilical cord mesenchymal stem cell confluence is 80-90%, the cell culture medium is replaced with DMEM / F12 medium without fetal bovine serum, 1% penicillin-streptomycin is added, and 1000 μM creatine is added for pretreatment for 12 hours.

[0054] Step 2: Use 0.25% trypsin (containing 0.01% EDTA) to digest the cells and add PBS to wash them.

[0055] Step 3: Except for the control group, the rest of the mice were smeared with R848 for 4 weeks to establish the model. The mice in the MSC treatment group were infused with 5×10 5 MSC cell number.

[0056] The results are as follows Figure 3 shown. Figure 3 The results of A showed that when Balb / c mice were induced into lupus model using R848, the IFN-γ level in the mouse serum increased significantly. The results of Figure 3B showed that the spleen of mice in the Cr pre-MSC group was significantly reduced, and it was more significant than that in the MSC group and the Cr pre-shSLC6A8-MSC group. The spleen weight index was also significantly reduced, while the spleen in the Cr group did not shrink significantly. The results of Figure 3C showed that the level of autoantibodies in the serum of the Cr pre-MSC group was also significantly reduced, and the changes were more obvious than those in the MSC group, the Cr pre-shSLC6A8-MSC group and the Cr group. Figure 3 D The results showed that in terms of renal function, both the MSC group and the Cr pre-MSC group significantly reduced the level of urine protein. Although there was no significant difference between the two groups, the Cr pre-MSC group tended to have lower urine protein than the MSC group. Figure 3E The results showed that the renal pathology was consistent with the changes in urine protein, indicating that the MSC group and the Cr pre-MSC group could significantly reduce the thickening of the renal basement membrane and the infiltration of inflammatory cells. No significant improvement was observed in the kidneys in the Cr pre-shSLC6A8-MSC group and the Cr group. The results of Figures 3F-3G showed that MSC treated with creatine also had a good regulatory effect on T lymphocyte subsets in vivo. The flow cytometry results showed that the proportions of CD4, CD8 naive T cells, and Tregs cells in the spleen of lupus mice were significantly increased after treatment with the Cr pre-MSC group, and there was a difference with the MSC group. Although there was no significant difference between the CD4 and CD8 Teff groups and the MSC group, there was also a significant downward trend compared with them.

[0057] The above results show that the pretreatment method of the present invention can improve the phenotype of lupus model and increase the proportion of CD4, CD8 naive T cells and Tregs cells in the spleen in lupus mouse model. The obtained MSCs can be used to prepare mesenchymal stem cell preparations and drugs to treat systemic lupus erythematosus.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: S1. Cell isolation: Take 5-10 cm of the umbilical cord of a newborn, cut it and immediately place it in sterile DMEM / F12 culture medium; Use PBS to wash away residual blood and clots on the surface of the umbilical cord, cut the umbilical cord into small pieces of 2-3 cm in a clean bench, remove the arteries and veins on the umbilical cord, wash it again with PBS, and peel off Wharton's jelly; S2. Cell culture: Cut Wharton's jelly into 2 mm pieces 3 Small pieces of MSC were spread on the bottom of the culture dish, and DMEM / F12 medium containing 10% fetal bovine serum by mass was added to the medium. Cell culture was carried out at 37°C, and fresh medium was replaced every 72-96 hours. After 14-21 days, primary cells were observed to grow around the tissue under a microscope and form colonies. They were digested with trypsin solution for 2 minutes and subcultured. When the cell fusion reached 80%, subculture was carried out again until the 6th generation of MSC was cultured. S3. Cell activation: When the cell confluence is 80-90%, use DMEM / F12 medium without fetal bovine serum to culture the cells, add 1% by volume of penicillin-streptomycin for screening, add creatine for pretreatment, and use IFN-γ cytokine to activate for 12 hours to obtain activated umbilical cord mesenchymal stem cells.

2. A pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The pancreatic enzyme solution in step S2 is a pancreatic enzyme solution with a mass percentage of 0.25%, including 0.01 mass% EDTA.

3. A pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The creatine pretreatment and IFN-γ cytokine activation treatment in step S3 can produce an additive effect.

4. An application of a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells, characterized in that: The mesenchymal stem cells prepared by the pretreatment method are used to prepare a mesenchymal stem cell preparation.

5. The use of a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells according to claim 4, characterized in that: The mesenchymal stem cells prepared by the pretreatment method are used for preparing medicines.

6. The use of a pretreatment method for increasing the immunoregulatory function of umbilical cord mesenchymal stem cells according to claim 4, characterized in that: The mesenchymal stem cells prepared by the pretreatment method are used for treating systemic lupus erythematosus.

7. The use according to claims 4-6, characterized in that: The IFN-γ cytokine in the pretreatment method can respectively increase the mRNA expression levels of COX2 and IDO-1 in umbilical cord mesenchymal stem cells, and the co-treatment of creatine and IFN-γ cytokine can further increase the mRNA expression levels of COX2, IL-10 and IDO in umbilical cord mesenchymal stem cells based on the level of IFN-γ activation.

8. The use according to claims 4-6, characterized in that: The pretreatment method can improve the proliferation inhibition function of umbilical cord mesenchymal stem cells on CD4 and CD8 T cells.

9. The use according to claims 4-6, characterized in that: The pretreatment method can regulate the proportion of T cell subsets in PBMC.

10. The use according to claims 4-6, characterized in that: The pretreatment method can improve the phenotype of the lupus model and increase the proportion of CD4, CD8 naive T cells and Tregs cells in the spleen in the lupus mouse model.

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