A method for culturing MSCs with enhanced immunosuppressive activity and its application in treatment of systemic lupus erythematosus

Abstract

This application belongs to the field of stem cell technology and biomedicine, specifically providing a method for culturing MSCs with enhanced immunosuppressive activity and its application in the treatment of systemic lupus erythematosus (SLE). The method for culturing MSCs with enhanced immunosuppressive activity includes the following steps: S1: Mesenchymal stem cells are harvested, expanded, and then pretreated with hypoxia; S2: The pretreated mesenchymal stem cells are sequentially induced with TL-4 and TGF-β1. This application, through a specific hypoxic pretreatment environment combined with a sequential induction process using immunomodulatory factors, regulates the metabolic patterns and signal transduction pathways of mesenchymal stem cells, thereby improving their survival rate, homing ability, and the level of immunosuppressive factor secretion in the complex immune microenvironment of SLE.

Description

Technical Field

[0001] This application belongs to the field of stem cell technology and biomedicine, and in particular relates to a method for culturing MSCs that enhances immunosuppressive activity and its application in the treatment of systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organs, characterized by abnormal T / B cell activation, massive production of autoantibodies, immune complex deposition, and organ damage. Lupus nephritis is one of its most common and serious complications. Mesenchymal stem cells (MSCs) possess powerful immunomodulatory, anti-inflammatory, and tissue repair functions, and have become an important research direction for SLE treatment.

[0003] In clinical applications, conventionally cultured MSCs have room for improvement in immunosuppressive activity, leading to inconsistent therapeutic efficacy in the complex in vivo microenvironment. Furthermore, the survival time of MSCs in vivo, their homing ability, and the functional consistency between different batches are all key factors affecting their standardized application. Current technologies often employ single hypoxia or single cytokine pretreatment; however, these methods require further optimization in generating synergistic effects and significantly enhancing cell function, thus limiting the ability of prepared MSC formulations to meet the demands for high-efficiency and stable clinical outcomes.

[0004] Patent CN115011553A discloses a method for preparing and using bone marrow mesenchymal stem cells derived from the neural crest of the trunk. This method involves inducing pluripotent stem cells and using a specific induction culture medium to define their differentiation pathway, successively undergoing neural mesodermal progenitor cell and neural crest cell stages to obtain MSCs. While this technology reduces cell heterogeneity and enhances immunomodulatory capabilities by clearly defining the cell source and differentiation pathway, it primarily focuses on standardizing the cell source through developmental biology pathways. In the specific pathological environment of systemic lupus erythematosus (SLE), characterized by strong inflammation and high immune response, this approach offers a relatively limited method for significantly enhancing immunosuppressive activity and prolonging in vivo survival time through a composite culture process.

[0005] Patent CN120718847A discloses a culture medium composition and a method for preparing mesenchymal stem cells (MSCs). The culture medium composition includes a three-stage culture medium for inducing pluripotent stem cells (ES or iPSCs) to prepare MSCs through a combination of small molecule compounds. This method solves the problems of large-scale MSC sourcing and standardization of the preparation process, making it applicable to the field of immunotherapy. However, this method mainly focuses on the efficiency of cell induction and transformation. It lacks in-depth microenvironment regulation methods for specifically improving the targeted homing ability of MSCs in the treatment of systemic lupus erythematosus and their functional persistence in a harsh immune microenvironment. Furthermore, the extent to which its immunosuppressive activity can be enhanced still shows certain limitations when dealing with complex autoimmune diseases. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a method for culturing MSCs that enhances immunosuppressive activity and its application in the treatment of systemic lupus erythematosus (SLE). The aim is to regulate the metabolic patterns and signal transduction pathways of mesenchymal stem cells (MSCs) through a specific hypoxic pretreatment environment combined with a sequential induction process of immunomodulatory factors, thereby improving their survival rate, homing ability, and the level of immunosuppressive factors secreted in the complex immune microenvironment of SLE.

[0007] This application first provides a method for culturing MSCs to enhance immunosuppressive activity, comprising the following steps: S1: Mesenchymal stem cells were harvested, expanded, and then subjected to hypoxia pretreatment. S2: Mesenchymal stem cells pretreated with hypoxia were sequentially induced and cultured using TL-4 and TGF-β1.

[0008] Furthermore, the amplification is performed by conventionally culturing cells to a degree of confluence of 70%-90%.

[0009] Furthermore, the oxygen concentration of the low-oxygen pretreatment is 1%-5%, and the treatment time is 12-48 hours; preferably, the oxygen concentration of the low-oxygen pretreatment is 2.5%, and the treatment time is 24 hours.

[0010] Furthermore, the TL-4 concentration in the sequential induction culture is 5-25 ng / mL, the TGF-β1 concentration is 2-15 ng / mL, and the induction time is 24-72 h; preferably, the TL-4 concentration in the sequential induction culture is 15 ng / mL, the TGF-β1 concentration is 8 ng / mL, and the induction time is 48 h.

[0011] Furthermore, the sequential induction culture is carried out under normoxic conditions.

[0012] Furthermore, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

[0013] This application provides a mesenchymal stem cell with high immunosuppressive activity obtained by the above method.

[0014] This application provides an application of the above-mentioned mesenchymal stem cells in systemic lupus erythematosus, wherein the mesenchymal stem cells are used to prepare a drug for treating systemic lupus erythematosus.

[0015] Furthermore, the drug is in the form of an injection and is administered intravenously.

[0016] Furthermore, this is manifested in improvements in one or more of the following indicators: a) Reduce anti-dsDNA antibody levels; b) Increase complement C3 and / or complement C4 levels; c) Reduce 24-hour urinary protein levels; d) Reduce disease activity scores; e) Reduce pathological damage to the kidneys.

[0017] Compared with the prior art, this application has the following beneficial effects: 1. The culture method described in this application significantly upregulates the secretion of key immunosuppressive factors such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1) by MSCs through the sequential synergistic effect of hypoxia and cytokines. Experiments show that compared with conventionally cultured MSCs, the MSCs of this application exhibit a 2.3-fold increase in IDO secretion, a 2.4-fold increase in PGE2, a 2.8-fold increase in IL-10, and a 2.4-fold increase in TGF-β1. Simultaneously, their inhibitory ability against pathogenic Th17 cells and their ability to induce regulatory Treg cells are greatly enhanced.

[0018] 2. In the classic MRL / lpr lupus mouse model, the MSCs prepared in this application significantly reduced serum anti-dsDNA antibody levels, increased complement C3 levels, reduced 24-hour urinary protein, decreased disease activity index (SLEDAI) scores, and effectively alleviated glomerular and tubular damage and inflammatory cell infiltration. All efficacy indicators were significantly superior to those of the conventional MSC treatment group.

[0019] 3. MSCs cultured using the method described in this application exhibit surface markers that meet international standards, and show no abnormal differentiation or tumorigenicity. No adverse reactions were observed in animal experiments, and the clinical application demonstrates good safety. Furthermore, the culture method described in this application utilizes a serum-free culture system with clearly defined and controllable conditions, and the sequential cytokine induction process is easy to standardize, providing a reliable solution for large-scale production that meets GMP requirements. Detailed Implementation

[0020] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] When using “including,” “having,” and “contains” as described herein, the intention is to cover non-exclusive inclusion, unless an explicit qualifying term such as “only,” “consisting of,” etc., is used, in which case another component may be added.

[0023] The terms "preferred," "more preferably," "better," and "even better" used in this application refer to embodiments of this application that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this application. That is, in this application, "preferred," "more preferably," "better," and "even better" are merely descriptions of implementations or embodiments with better effects, but do not constitute a limitation on the scope of protection of this application.

[0024] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0025] In this application, "at least one" means one or more, such as one, two, or more. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layered" means at least two layers, such as two layers, three layers, etc., unless otherwise explicitly specified. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0026] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0027] Unless otherwise specified, all steps in this application may be performed sequentially or randomly. For example, the method comprising steps (a) and (b) indicates that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc. Unless otherwise stated, singular terms may include plural forms and should not be construed as having a quantity of one.

[0028] In this application, "above" or "below" includes the number itself. For example, "below 1" includes 1.

[0029] In this application, room temperature refers to 0~40℃, including but not limited to 10~40℃, or further to 20~30℃.

[0030] Based on extensive experimental research, this application provides a method for culturing MSCs that enhances immunosuppressive activity, comprising the following steps: S1: Mesenchymal stem cells were harvested, expanded, and then subjected to hypoxia pretreatment. S2: Mesenchymal stem cells pretreated with hypoxia were sequentially induced and cultured using TL-4 and TGF-β1.

[0031] Furthermore, the amplification is performed by conventionally culturing cells to a degree of confluence of 70%-90%.

[0032] In some specific embodiments, the amplification is carried out by conventional culture until the cell confluence reaches 70%-75%, 75%-80%, 80%-85%, or 85%-90%. Under normal circumstances, the amplification is carried out by conventional culture until the cell confluence reaches 85%-90%, which can achieve better technical results.

[0033] Furthermore, the oxygen concentration of the low-oxygen pretreatment is 1%-5%, and the treatment time is 12-48 hours; preferably, the oxygen concentration of the low-oxygen pretreatment is 2.5%, and the treatment time is 24 hours.

[0034] In some specific implementations, the oxygen concentration of the hypoxia pretreatment is 2.5%, the carbon dioxide concentration is 5%, and the treatment time is 24 hours, which can achieve better experimental results.

[0035] Furthermore, the TL-4 concentration in the sequential induction culture is 5-25 ng / mL, the TGF-β1 concentration is 2-15 ng / mL, and the induction time is 24-72 h; preferably, the TL-4 concentration in the sequential induction culture is 15 ng / mL, the TGF-β1 concentration is 8 ng / mL, and the induction time is 48 h.

[0036] In some embodiments, the sequential induction culture uses a complete culture medium.

[0037] Furthermore, the sequential induction culture is carried out under normoxic conditions.

[0038] Furthermore, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

[0039] This application provides a mesenchymal stem cell with high immunosuppressive activity obtained by the above method.

[0040] This application provides an application of the above-mentioned mesenchymal stem cells in systemic lupus erythematosus, wherein the mesenchymal stem cells are used to prepare a drug for treating systemic lupus erythematosus.

[0041] In some embodiments, the systemic lupus erythematosus includes lupus nephritis.

[0042] Furthermore, the drug is in the form of an injection and is administered intravenously.

[0043] In some specific embodiments, the injectable preparation has the active ingredient being the MSC that enhances immunosuppressive activity, and the solvent can be physiological saline containing 1%-5% human serum albumin by mass.

[0044] In some embodiments, the drug is administered via intravenous infusion or intravenous injection, and the dosage can be 1.0 × 10⁻⁶ per kilogram of body weight. 6 -2.0×10 6 MSCs are administered once every 7-14 days for 3 to 5 consecutive times.

[0045] This application provides a composite induction culture medium for enhancing the immunosuppressive activity of MSCs, comprising a basal culture medium and additive components, wherein the additive components include: IL-4 at a concentration of 10-20 ng / mL; and TGF-β1 at a concentration of 5-15 ng / mL.

[0046] This application provides a combination therapy regimen for treating systemic lupus erythematosus, comprising administering the aforementioned MSC preparation with enhanced immunosuppressive activity to the patient, and concurrently administering a low dose of glucocorticoids. The dose of the glucocorticoids may be 0.1-0.5 mg / kg daily.

[0047] Furthermore, this is manifested in improvements in one or more of the following indicators: a) Reduce anti-dsDNA antibody levels; b) Increase complement C3 and / or complement C4 levels; c) Reduce 24-hour urinary protein levels; d) Reduce disease activity scores; e) Reduce pathological damage to the kidneys.

[0048] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0049] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, or made by the methods, equipment, and materials in the embodiments of this application may be used to implement this application.

[0050] Preparation Example The method for isolating and culturing human umbilical cord MSCs in this preparation example includes the following steps: Healthy full-term cesarean section umbilical cords were collected, aseptically minced to 1 mm³ tissue blocks, and 0.1% collagenase IV was added. The cells were digested at 37°C for 60 min. After digestion was terminated, the cells were filtered and centrifuged. The centrifuged cells were resuspended in serum-free Dakota medium, seeded in T75 culture flasks, and cultured at 37°C and 5% CO2. The medium was changed every 3 days. When the cells reached 85% confluence, they were digested with 0.25% trypsin and passaged to obtain P3 generation MSCs.

[0051] The seed cell culture process for this preparation example is shown in Table 1.

[0052] Table 1 Seed cell culture process of the preparation example in this application Example The MSC culture method for enhancing immunosuppressive activity in this embodiment includes the following steps: S1: P3 generation MSCs were amplified at a rate of 2.0 × 10⁶ cells / T175 and cultured until 85%–90% confluence. After amplification, hypoxia pretreatment was performed at an oxygen concentration of 2.5%, a carbon dioxide concentration of 5%, a temperature of 37°C, and a treatment time of 24 h. S2: Mesenchymal stem cells pretreated with hypoxia were sequentially induced with TL-4 and TGF-β1. The induction culture was carried out in a complete medium containing 15 ng / mL IL-4 + 8 ng / mL TGF-β1. The induction was carried out under normoxic conditions for 48 h. After induction, the cells were washed twice with PBS, digested with trypsin, centrifuged and resuspended for later use.

[0053] control group The MSC culture method in this control group included the following steps: S1: P3 generation MSCs were amplified at a seeding rate of 2.0 × 10⁶ cells / T175 and cultured until 85%–90% confluence. After amplification, routine pretreatment was performed at a carbon dioxide concentration of 5% and a temperature of 37°C for 24 hours. S2: Mesenchymal stem cells that have undergone routine pretreatment were sequentially induced with TL-4 and TGF-β1. The induction culture was carried out in a complete medium containing 15 ng / mL IL-4 + 8 ng / mL TGF-β1. The induction was carried out under normoxic conditions for 48 h. After the induction was completed, the cells were washed twice with PBS, digested with trypsin, centrifuged and resuspended for later use.

[0054] Table 2 shows a comparison of the cell culture processes of the embodiments and the control group in this application.

[0055] Table 2 Comparison of cell culture processes between the embodiments of this application and the control group Performance testing I. Quality Assay of P4 Generation MSCs: 1. Phenotypic detection, the results are shown in Table 3.

[0056] Table 3. Phenotypic detection of the embodiments and control group in this application. Example: CD73=98.7%, CD90=99.1%, CD105=97.8%; CD34 / CD45 / HLA-DR are all <1.5%.

[0057] Control group: CD73=96.2%, CD90=97.5%, CD105=95.3%.

[0058] 2. Immunosuppressive factor secretion (ELISA) results are shown in Table 4.

[0059] Table 4. Secretion of immunosuppressive factors in the embodiments of this application and the control group. IDO: Example 32.6 ng / mL, control group 14.2 ng / mL (2.3-fold increase) PGE2: Example 8 95.3 pg / mL, control group 368.7 pg / mL (2.4-fold increase) IL-10: Example 1: 28.6 pg / mL, Control group: 45.2 pg / mL (2.8-fold increase) TGF-β1: Example 2: 15.4 pg / mL, control group: 89.7 pg / mL (2.4-fold increase) 3. Immunomodulatory function: The culture method in this application embodiment can increase the proportion of Treg from 4.2% to 15.7% and decrease the proportion of Th17 from 9.8% to 2.1%, thereby increasing the inhibition efficiency by 68%.

[0060] II. SLE Animal Model Therapy Experiment: 1. Animal grouping: MRL / lpr lupus mice were randomly divided into 3 groups (n=10). Model group: physiological saline Conventional MSC group: Control group MSCs cultured 5 × 10 5 cells / only This application group: Example: Culture of MSCs 5 × 10 5 cells / only 2. Administration method: Inject via tail vein, once every 7 days, for a total of 4 times.

[0061] 3. Detection Indicators and Results The specific test results are shown in Table 5.

[0062] Table 5 Results of experimental detection indicators of SLE animal model efficacy in this application group, conventional group and this application group 1) Anti-dsDNA antibody Model group: 852.6 IU / mL; Conventional group: 518.3 IU / mL; Group specified in this application: 226.7 IU / mL 2) Complement C3 Model group: 0.18 g / L; Conventional group: 0.32 g / L; Group specified in this application: 0.57 g / L 3) 24-hour urine protein Model group: 12.8 mg / 24h; Conventional group: 7.3 mg / 24h; Group specified in this application: 2.9 mg / 24h 4) SLEDAI score Model group: 14.8 points; Conventional group: 9.3 points; This application group: 4.1 points 5) Kidney pathology score The specific scoring results are shown in Table 6.

[0063] Table 6. Renal pathology scores of the model group, conventional group, and the group in this application. Model group: 12.6 points; Conventional group: 8.2 points; This application group: 3.5 points Conclusion: The MSCs prepared in this application have a significantly better therapeutic effect on SLE than conventionally cultured MSCs.

[0064] III. Safety Testing 1. Negative for mycoplasma, bacteria, and fungi. 2. Endotoxin <0.5 EU / mL 3. Cells show no abnormal differentiation and no tumorigenicity. 4. No adverse reactions such as allergies, fever, or organ damage were observed in animal experiments. 5. Supporting experimental data To verify the non-obviousness and technical superiority of the hypoxia pretreatment combined with IL-4 and TGF-β1 sequential induction protocol described in this application in enhancing the immunosuppressive activity of MSCs and the therapeutic effect on SLE, this application set up multiple control experiments to systematically compare sequential induction with synchronous induction, single factor induction with composite factor induction, and hypoxia pretreatment with normoxic culture. The experimental results are as follows.

[0065] 5.1 Comparison Experiment of Sequential Induction and Synchronous Induction This experiment sets up two treatment methods: 5.1.1 Sequential induction group: MSCs were first pretreated with hypoxia, and then IL-4 and TGF-β1 were added sequentially for induction. 5.1.2 Synchronous Induction Group: MSCs were simultaneously treated with IL-4 and TGF-β1 under normoxic conditions.

[0066] The detection indicators included the expression levels of key MSC immunosuppressive molecules PD-L1, IDO, and HO-1, the T cell proliferation inhibition rate, and the in vivo treatment effect in SLE model mice.

[0067] Experimental results showed that the expression levels of PD-L1, IDO, and HO-1 in MSCs of the sequential induction group were significantly higher than those of the synchronous induction group, with an increase of 3.8 times; the inhibition rate of activated T cell proliferation increased from 41.2% in the synchronous group to 78.5%; in the SLE mouse model, the sequential induction group could significantly reduce serum anti-dsDNA antibody levels, improve renal pathological damage, and reduce the disease activity index (DAI), with significantly better therapeutic effects than the synchronous induction group.

[0068] The above results confirm that sequential treatment with IL-4 and TGF-β1 produces significantly better immunomodulatory effects than simultaneous treatment. This treatment method is not a conventional choice in the field and is therefore non-obvious.

[0069] 5.2 Comparative Experiment of Single-Factor Induction and Composite-Factor Induction This experiment was set up with four groups: blank control group, IL-4 alone induction group, TGF-β1 alone induction group, and IL-4+TGF-β1 combined sequential induction group.

[0070] The expression of immune regulation-related genes ARG1, TGF-β, and IL-10 and their in vitro immunosuppressive function were detected.

[0071] The experimental results showed that the single-factor treatment group could only slightly upregulate the expression of immunosuppression-related molecules, and the inhibition rate of T cell proliferation was less than 35%; while the IL-4 and TGF-β1 combined sequential induction group showed a significant synergistic effect, with the expression levels of genes such as ARG1 and IL-10 being higher than the sum of the single-factor groups, significantly improving the inhibition efficiency of autoreactive T cells, and significantly reducing the level of inflammatory factors in the SLE model.

[0072] The results show that the combination of IL-4 and TGF-β1 used in this application is not a simple superposition of the effects of a single factor, but produces an unexpected synergistic enhancement effect, demonstrating outstanding technological innovation.

[0073] 5.3 Validation of the synergistic effect of hypoxia pretreatment combined with sequential complex factor combination This application further compares the following groups: normoxic culture group, hypoxic pretreatment group, IL-4+TGF-β1 sequential group, and hypoxic pretreatment + IL-4+TGF-β1 sequential combination group.

[0074] Experimental results showed that neither hypoxia pretreatment alone nor factor induction alone could achieve the desired immunosuppressive effect; however, hypoxia pretreatment combined with sequential complex factor induction could further activate the HIF-1α pathway, synergistically upregulate the immunosuppressive function of MSCs, significantly improve the cells' tolerance and repair capacity to the SLE-related inflammatory microenvironment, and achieve a more stable and longer-lasting disease remission effect in animal models.

[0075] The above data confirms that the overall technical solution of hypoxia pretreatment + specific sequential complex factor in this application has no relevant inspiration from the prior art in terms of enhancing MSC immunosuppressive activity and treating SLE, and has outstanding non-obviousness and significant progress.

[0076] 6. SLE-specific efficacy data Highly active MSCs prepared according to the embodiments of this application were used to treat SLE model mice, and the core pathological markers of SLE were detected. 6.1 Anti-dsDNA antibody titer: decreased by 68.2% compared to the model group; 6.2 Complement C3 / C4 levels: Significantly increased and approaching normal levels; 6.3 Renal pathological damage: glomerular immune complex deposition, renal tubular damage, and inflammatory cell infiltration were significantly reduced; 6.4 Inflammatory infiltration of kidney tissue: The number of CD4+ T cells and macrophages decreased by more than 70%; 6.5 Disease Activity Index (DAI): Significantly lower than the control group.

[0077] Data shows that the MSCs prepared by the culture method of this application have a specific and significant therapeutic effect on SLE. This effect has no clear inspiration in the prior art and has outstanding non-obviousness and significant progress.

[0078] In summary: This application discloses a method for culturing MSCs with enhanced immunosuppressive activity and its application in the treatment of systemic lupus erythematosus (SLE). This method employs 2.5% hypoxia pretreatment for 24 hours combined with sequential induction by IL-4 and TGF-β1, significantly increasing the secretion of immunosuppressive factors by MSCs and enhancing their ability to correct Th17 / Treg immune imbalance. Experiments show that these MSCs can significantly reduce anti-dsDNA antibodies, increase complement C3 / C4 ratios, reduce proteinuria, alleviate lupus nephritis damage, and significantly improve disease activity in SLE model mice. The process described in this application is stable and can be mass-produced, providing a highly efficient and safe novel stem cell therapy for refractory SLE.

[0079] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for culturing MSCs to enhance immunosuppressive activity, characterized by: Includes the following steps: S1: Mesenchymal stem cells were harvested, expanded, and then subjected to hypoxia pretreatment. S2: Mesenchymal stem cells pretreated with hypoxia were sequentially induced and cultured using TL-4 and TGF-β1.

2. The MSC culture method for enhancing immunosuppressive activity according to claim 1, characterized by: The amplification is performed by conventionally culturing cells until the cell confluence reaches 70%-90%.

3. The MSC culture method for enhancing immunosuppressive activity according to claim 1, characterized by: The oxygen concentration for the low-oxygen pretreatment is 1%-5%, and the treatment time is 12-48 hours; preferably, the oxygen concentration for the low-oxygen pretreatment is 2.5%, and the treatment time is 24 hours.

4. The MSC culture method for enhancing immunosuppressive activity according to claim 1, characterized by: The sequential induction culture uses a TL-4 concentration of 5-25 ng / mL, a TGF-β1 concentration of 2-15 ng / mL, and an induction time of 24-72 h; preferably, the sequential induction culture uses a TL-4 concentration of 15 ng / mL, a TGF-β1 concentration of 8 ng / mL, and an induction time of 48 h.

5. The MSC culture method for enhancing immunosuppressive activity according to claim 1, characterized by: The sequential induction culture was carried out under normoxic conditions.

6. The MSC culture method for enhancing immunosuppressive activity according to claim 1, characterized in that: The mesenchymal stem cells mentioned are human umbilical cord-derived mesenchymal stem cells.

7. A mesenchymal stem cell with high immunosuppressive activity obtained by the method described in any one of claims 1-6.

8. An application of mesenchymal stem cells as described in claim 7 in systemic lupus erythematosus, characterized in that: The mesenchymal stem cells were used to prepare a drug for treating systemic lupus erythematosus.

9. The application according to claim 8, characterized in that: The drug is in the form of an injection and is administered intravenously.

10. The application according to claim 8, characterized in that: Improvement is manifested in one or more of the following indicators: a) Reduce anti-dsDNA antibody levels; b) Increase complement C3 and / or complement C4 levels; c) Reduce 24-hour urinary protein levels; d) Reduce disease activity scores; e) Reduce pathological damage to the kidneys.

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