Preparation method and application of IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregate

Abstract

The invention discloses an IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregate preparation method and application, and the method comprises the following steps: using IL-6 to pre-stimulate umbilical cord mesenchymal stem cells to obtain pre-stimulated cells, then adding the pre-stimulated cells into an Aggrewell 400 culture plate for centrifugation, continuing to culture the centrifuged pre-stimulated cells for 12-16 h, and obtaining the IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregate. The pre-stimulated mesenchymal stem cell aggregate is obtained, and the umbilical cord mesenchymal stem cells are in-vitro cells. The invention provides a method for pre-stimulating an umbilical cord mesenchymal stem cell aggregate by using IL-6, which can improve the expression level of inhibitory factors, inhibit the proliferation ability of human peripheral blood mononuclear cells and inhibit the proliferation ability of human CD4 positive T lymphocytes. Through the spherical three-dimensional aggregate structure formed by mutual adhesion of mesenchymal stem cells, the microenvironment of cells in vivo can be better simulated, the survival rate of transplanted stem cells is increased, the retention time in vivo is prolonged, and the treatment effect is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates. Background Art

[0002] Systemic sclerosis (SSc), also known as scleroderma, is a rare autoimmune disease characterized by localized or diffuse skin thickening and hardening and tissue fibrosis. Conventional treatments are mainly aimed at immune disorders, vascular lesions and abnormal collagen deposition, with immune regulation, microcirculation improvement and fibrosis reduction as the basis of treatment. Conventional treatment drugs include glucocorticoids, immunosuppressants, penicillamine and methotrexate, but the above drugs are not only non-specific in inhibiting the proliferation and activity of immune cells, but also have the risk of causing serious complications such as bone marrow suppression, infection, liver and kidney damage.

[0003] CN115418346 discloses a preparation method for adding interferon gamma and tumor necrosis factor alpha pre-stimulation to the existing mesenchymal stem cell culture model, which greatly increases the content of anti-inflammatory and immunomodulatory factors in the cell supernatant. Selecting appropriate inflammatory factor pre-stimulation for different diseases may help mesenchymal stem cells to be reactivated by highly expressed inflammatory factors in the body after infusion. Given the important role of interleukin 6 (IL-6) in the pathogenesis of scleroderma, and the fact that tocilizumab targeting IL-6 is the only biological agent approved for the treatment of systemic sclerosis-related interstitial lung disease, the use of IL-6 may be more suitable than other inflammatory factors for pre-stimulation of mesenchymal stem cells in the context of systemic sclerosis.

[0004] At present, most mesenchymal stem cells used to treat systemic sclerosis are cultured using the traditional adherent method, which has the following limitations: (1) Mesenchymal stem cells cultured using the traditional adherent method have a short residence time in the body; (2) Mesenchymal stem cells cultured using the traditional adherent method cannot simulate the real environment of stem cells in the body, and lack sufficiently tight and complex cell connections between cells, resulting in restrictions on their biological functions such as proliferation, migration, and differentiation. They cannot adapt to the harsh microenvironment after transplantation and die prematurely, losing their therapeutic ability; (3) Mesenchymal stem cells cultured using traditional methods lack the induction of inflammatory response / or other stimuli, their immunoregulatory properties have not been remodeled, and their immunosuppressive ability has not been fully activated. (4) There is currently no inflammatory factor specifically designed to target the pathogenesis and characteristics of systemic sclerosis and to consider the most suitable inflammatory factor for pre-stimulating mesenchymal stem cells under the pathological state of the disease. Summary of the invention

[0005] Based on this, the present invention provides a preparation method and application of IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates, which better simulate the microenvironment of cells in the body through the spherical three-dimensional aggregate structure formed by mutual adhesion between mesenchymal stem cells, enhance the survival rate, in vivo retention time and therapeutic effect of stem cells after transplantation, and use IL-6 pre-stimulation to enhance the immunosuppressive ability of mesenchymal stem cells. The specific scheme is as follows: On the one hand, the present invention provides a method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates, comprising the following steps: using IL-6 to pre-stimulate umbilical cord mesenchymal stem cells to obtain pre-stimulated cells, then adding the pre-stimulated cells to an Aggrewell 400 culture plate for centrifugation, and continuing to culture the centrifuged pre-stimulated cells for 12-16 hours to obtain pre-stimulated cell aggregates, wherein the umbilical cord mesenchymal stem cells are in vitro cells.

[0006] Preferably, the IL-6 concentration is 10 ng / ml, 20 ng / ml, 40 ng / ml and 80 ng / ml.

[0007] Preferably, the centrifugation condition of the culture plate is 1000 rpm, 5 min.

[0008] Preferably, the pre-stimulated cells have at least one of the following characteristics A1)-A3): A1) the expression level of immunosuppressive factors in the pre-stimulated cells is higher than that in the umbilical cord mesenchymal stem cells; A2) the ability of the pre-stimulated cells to inhibit the proliferation of human peripheral blood mononuclear cells is higher than that of the umbilical cord mesenchymal stem cells; A3) The ability of the pre-stimulated cells to inhibit the proliferation of human CD4-positive T lymphocytes is higher than that of the umbilical cord mesenchymal stem cells.

[0009] Preferably, the immunosuppressive factor is one or more of IDO, HGF, COX-2, and TSG6.

[0010] Preferably, the proliferation of the human peripheral blood mononuclear cells and CD4-positive T lymphocytes is detected by CFSE staining.

[0011] In a second aspect, the present invention provides a use of pre-stimulated cell aggregates in the preparation of immunosuppressive products, and the use of the pre-stimulated cell aggregates in drugs and / or vaccines.

[0012] In a third aspect, the present invention provides an application of pre-stimulated cell aggregates in the preparation of products for reducing inflammatory damage, and the application of the pre-stimulated cell aggregates in drugs and / or vaccines.

[0013] Beneficial effects: The present invention provides a preparation method and application of IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates, and its beneficial effects are as follows: 1. Enhance immunosuppressive ability: Pre-stimulation of umbilical cord mesenchymal stem cell aggregates with IL-6 can increase the expression level of inhibitory factors (such as IDO, HGF, COX-2, TSG6, etc.). Experiments have shown that under the stimulation of 40ng / ml IL-6, the expression of immunosuppressive factors in mesenchymal stem cells increased most significantly, such as IDO expression level can reach 1.99-2.79 times that of unstimulated, HGF is 2.08-2.18 times, COX-2 is 4.3-8.2 times, TSG6 is 5.3-8.2 times.

[0014] The ability of pre-stimulated cells to inhibit the proliferation of human peripheral blood mononuclear cells and human CD4-positive T lymphocytes was significantly higher than that of untreated umbilical cord mesenchymal stem cells. In the cell proliferation inhibition experiment, the ability of pre-stimulated mesenchymal stem cell aggregates to inhibit the proliferation of PBMC and CD4-positive T lymphocytes was significantly improved, which helps to regulate the body's immune response and is of great significance in the treatment of autoimmune diseases.

[0015] 2. Optimize cell microenvironment simulation and transplantation effect: The spherical three-dimensional aggregate structure formed by the mutual adhesion of mesenchymal stem cells better simulates the microenvironment of cells in the body. This structure makes the connection between cells closer. For example, during the culture process, the pre-stimulated mesenchymal stem cell aggregates shrink spontaneously over time and become smaller in size, indicating that the connection between cells is continuously optimized.

[0016] It enhances the survival rate, in vivo retention time and therapeutic effect of transplanted stem cells. In the bleomycin-induced scleroderma mouse model treatment experiment, the mice in the pre-stimulated MSCs aggregate treatment group had the least skin fibrosis, the least inflammatory cell infiltration and capillary loss in the dermis, and could inhibit the differentiation of Th1 and Th17 cells to the greatest extent, indicating that its therapeutic effect in vivo is significantly better than that of mesenchymal stem cells cultured by traditional methods, providing a more effective means for the treatment of diseases such as systemic sclerosis.

[0017] 3. Providing targeted treatment strategies: Considering the important role of interleukin 6 (IL-6) in the pathogenesis of scleroderma, and the application of tocilizumab targeting IL-6 in the treatment of systemic sclerosis-related interstitial lung disease, the present invention uses IL-6 to pre-stimulate mesenchymal stem cells, providing a more targeted treatment strategy for diseases such as systemic sclerosis, which is expected to overcome the non-specificity and serious complications of traditional therapeutic drugs.

[0018] 4. Promote the development of stem cell therapy technology: This invention provides a new method and idea for the pre-stimulation and application of mesenchymal stem cells, which will help promote the further development of stem cell therapy technology in the field of biomedicine and provide reference and reference for the treatment of other similar diseases. It has important scientific significance and potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression of immunosuppressive factors in mesenchymal stem cells cultured by the traditional adherent method under the stimulation of different concentrations of IL-6 in Example 1 of the present invention.

[0020] Figure 2 This is a bright field observation picture of the aggregate in Example 2 of the present invention.

[0021] Figure 3 These are flow cytometer detection diagrams of PBMC proliferation in Example 3 of the present invention: A is a flow cytometer detection result diagram of inhibiting PBMC proliferation using the control group, activation group, MSCs group, and pre-stimulated MSCs aggregate group respectively; B is a statistical diagram of the results of A.

[0022] Figure 4 These are flow cytometer detection diagrams of human CD4-positive T lymphocyte proliferation in Example 3 of the present invention: A is a flow cytometer diagram of the inhibition of human CD4-positive T lymphocyte proliferation using the control group, activation group, MSCs group, and pre-stimulated MSCs aggregate group respectively; B is a statistical diagram of the results of A.

[0023] Figure 5 This is the HE staining image of the skin in Example 4 of the present invention.

[0024] Figure 6 This is the skin Masson staining diagram of Example 4 of the present invention.

[0025] Figure 7 Flow cytometry and statistical graphs of spleen type 1 T helper cells in Example 4 of the present invention.

[0026] Figure 8 Flow cytometry and statistical diagram of spleen helper T cells 17 in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the implementation cases provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The embodiments provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0028] The human umbilical cord mesenchymal stem cells in the following examples are primary umbilical vein mesenchymal stem cells. The PBMCs in the following examples are primary cells.

[0029] The DMEM / F12 culture medium, penicillin-streptomycin mixed double antibody, and fetal bovine serum in the following examples are all from Nanjing Shenghang Biotechnology Co., Ltd.; human interleukin-6 recombinant protein is a product of Perotech; propylene glycol methyl ether acetate (PMA), ionnmycin, and Brefeldin A are all products of eBioscience; CFSE, anti-human CD3 monoclonal antibody, and anti-human CD28 monoclonal antibody are all products of Invitrogen; Aggrewell400 is a product of StemCell; TRIZOL, reverse transcription kit, and SYBR Green reagent are all products of Nanjing Novozymes.

[0030] The quantitative data in the following examples were analyzed using GraphPad 8.0 software, and the sample means were compared using one-way analysis of variance, with P < 0.05 indicating statistically significant differences.

[0031] Example 1: Selecting the optimal concentration of IL-6 to stimulate human umbilical cord mesenchymal stem cells In this example, primary human umbilical vein endothelial cells were used as recipient mesenchymal stem cells, and different concentrations of human IL-6 recombinant protein were used to stimulate the recipient mesenchymal stem cells, thereby obtaining mesenchymal stem cells with increased expression of immunosuppressive factors compared with the recipient mesenchymal stem cells, and thus selecting the optimal human IL-6 recombinant protein stimulation concentration.

[0032] The specific method is as follows: Fluorescence quantitative PCR was used to detect the gene expression levels of IDO, HGF, COX-2 and TSG6 in umbilical cord mesenchymal stem cells before and after pre-stimulation.

[0033] Culture medium N1: DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, 1% (V / V) penicillin-streptomycin mixed double antibody, and 0 ng / ml IL-6.

[0034] Culture medium N2: DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, 1% (V / V) penicillin-streptomycin mixed double antibody, and 10 ng / ml IL-6.

[0035] Culture medium N3: DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, 1% (V / V) penicillin-streptomycin mixed double antibody, and 20ng / ml IL-6.

[0036] Culture medium N4: DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, 1% (V / V) penicillin-streptomycin mixed double antibody, and 40ng / ml IL-6.

[0037] Culture medium N5: DMEM / F12 culture medium containing 10% (V / V) fetal bovine serum, 1% (V / V) penicillin-streptomycin mixed double antibody, and 80ng / ml IL-6.

[0038] Human umbilical vein endothelial cells were cultured separately with culture medium N (culture medium N was culture medium N1, culture medium N2, culture medium N3, culture medium N4 or culture medium N5) at a rate of 1.5×10 5 Pieces / cm 2 The cells were seeded in a 6-well plate at a density of 1.54 μg / mL and placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. Then, real-time fluorescence quantitative PCR was performed to detect the gene expression levels of IDO, HGF, COX-2 and TSG6 in the cells.

[0039] Total RNA of cells was extracted with TRIZOL and reverse transcribed into cDNA using a reverse transcription kit. cDNA was used as a template and SYBR Green reagent was added to determine the expression of IDO, HGF, COX-2 and TSG6 genes by real-time fluorescence quantitative PCR, expressed relative to the expression of GAPDH gene. The experiment was repeated three times.

[0040] Among them, human GAPDH gene primers: Forward: 5'-ACAACTTTGGTATCGTGGAAGG-3' (SEQ ID No.1); Reverse: 5'-GCCATCACGCCACAGTTTC-3' (SEQ ID No.2); Human IDO gene primers: Forward: 5'-TGGGGCAAAGGTCATGGAG-3' (SEQ ID No.3); Reverse: 5'-TTTCTTGGAGAGTTGGCAGTAAG-3' (SEQ ID No.4); Human HGF gene primers: Forward: 5'-GCTATCGGGGTAAAAGACCTACA-3' (SEQ ID No.5); Reverse: 5'-CGTAGCGTACCTCTGGATTGC-3' (SEQ ID No.6); Human COX-2 gene primers: Forward: 5'-TAAGTGCGATTGTACCCGGAC-3' (SEQ ID No.7); Reverse: 5'-TTTGTAGCCATAGTCAGCATTGT-3' (SEQ ID No.8); Human TSG6 gene primers: Forward: 5'-TCTGGCAAATACAAGCTCACC-3' (SEQ ID No.9); Reverse: 5'-CTGCCCTTAGCCATCCATCC-3' (SEQ ID No. 10).

[0041] The results are as follows Figure 1 As shown, for the same number of cells, under the treatment of 10 ng / ml IL-6, the IDO expression level of the pre-stimulated cells was 0.83-1.65 times that of the mesenchymal stem cells, the HGF expression level of the pre-stimulated cells was 1.14-1.72 times that of the mesenchymal stem cells, the COX-2 expression level of the pre-stimulated cells was 1.05-1.55 times that of the mesenchymal stem cells, and the TSG6 expression level of the pre-stimulated cells was 1.47-1.81 times that of the mesenchymal stem cells.

[0042] For the same number of cells, under the treatment of 20 ng / ml IL-6, the IDO expression level of the pre-stimulated cells is 1.34-1.54 times that of the mesenchymal stem cells, the HGF expression level of the pre-stimulated cells is 1.74-1.88 times that of the mesenchymal stem cells, the COX-2 expression level of the pre-stimulated cells is 1.18-3.39 times that of the mesenchymal stem cells, and the TSG6 expression level of the pre-stimulated cells is 1.41-2.5 times that of the mesenchymal stem cells.

[0043] For the same number of cells, under the treatment of 40ng / ml IL-6, the IDO expression level of the pre-stimulated cells is 1.99-2.79 times that of the mesenchymal stem cells, the HGF expression level of the pre-stimulated cells is 2.08-2.18 times that of the mesenchymal stem cells, the COX-2 expression level of the pre-stimulated cells is 4.3-8.2 times that of the mesenchymal stem cells, and the TSG6 expression level of the pre-stimulated cells is 5.3-8.2 times that of the mesenchymal stem cells.

[0044] For the same number of cells, under the treatment of 80 ng / ml IL-6, the IDO expression level of the pre-stimulated cells is 1.48-2.61 times that of the mesenchymal stem cells, the HGF expression level of the pre-stimulated cells is 1.14-1.75 times that of the mesenchymal stem cells, the COX-2 expression level of the pre-stimulated cells is 0.94-1.42 times that of the mesenchymal stem cells, and the TSG6 expression level of the pre-stimulated cells is 1.03-1.58 times that of the mesenchymal stem cells.

[0045] The above results show that under the stimulation of 40ng / ml IL-6, the expression of immunosuppressive factors in mesenchymal stem cells increased most significantly.

[0046] Example 2: Preparation of pre-stimulated mesenchymal stem cell aggregates This example provides a method for preparing pre-stimulated mesenchymal stem cell aggregates.

[0047] Umbilical cord mesenchymal stem cells pre-stimulated with 40 ng / ml IL-6 for 24 hours were cultured at 5×10 5 The cells were added to the Aggrewell 400 culture plate at a density of 500 / ml (500 / microwell), and then centrifuged at 1000 rpm for 5 minutes. The culture plate was then placed in a cell culture incubator at 37°C and incubated for 12 hours. The pre-stimulated cell aggregates were imaged using an optical microscope. Figure 2 It can be seen that as the culture time increases, the pre-stimulated mesenchymal stem cell aggregates shrink spontaneously and become smaller and smaller, indicating that the connection between cells is tighter. When cultured for 12 hours, the diameter of the pre-stimulated cell aggregates hardly changes. In subsequent embodiments of the present invention, the immunosuppressive ability of mesenchymal stem cells cultured by the traditional adherent method and pre-stimulated mesenchymal stem cell aggregates in vivo and in vitro is compared.

[0048] Example 3: Cell proliferation inhibition experiment This example demonstrates through in vitro experiments that pre-stimulated mesenchymal stem cell aggregates have a stronger ability to inhibit the proliferation of PBMC and CD4-positive T cells.

[0049] The mesenchymal stem cells cultured by the conventional adherence method and the pre-stimulated mesenchymal stem cell aggregates prepared in Example 2 were co-cultured with human PBMC for 3 days, and the proliferation of PBMC and CD4-positive T lymphocytes was detected by flow cytometry. PBMC was pre-labeled by CFSE method.

[0050] The specific method is as follows: After removing plasma from the whole blood, add pre-cooled PBS to make up to the volume of whole blood. After mixing, use a dropper to slowly superimpose the diluted blood on the surface of the Ficoll separation liquid along the tube wall at a volume ratio of 1:1. Centrifuge at 2000 rpm at room temperature for 20 minutes (acceleration level 5, reduction speed level 2). After centrifugation, the tube is divided into three layers, the upper layer is PBS diluted plasma, the middle layer is Ficoll, and the lower layer is mainly red blood cells and granulocytes. There is a narrow band of white cloud layer at the junction of the upper and middle layers, which is the mononuclear cell. Use a dropper to carefully absorb the cloud layer cells and transfer them to a new 15ml centrifuge tube. Add pre-cooled PBS to the centrifuge tube to a total volume of 14ml, centrifuge at 1800rpm for 5min, and the resulting precipitate is PBMC.

[0051] PBMCs were stained with CFSE and resuspended in PBS to a density of 5-10×10 6 / ml, add CFSE solution with a final concentration of 1 μM, mix well and stain at room temperature in the dark for 10 minutes. Use complete medium with 4-5 times the volume of staining solution to stop staining and wash 3 times.

[0052] 24 hours in advance, the mesenchymal stem cells cultured by the conventional method and the pre-stimulated mesenchymal stem cell aggregates prepared in Example 2 were respectively cultured at 2×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate. CFSE-labeled PBMCs were seeded in the 96-well plate and divided into the following groups: (1) control group; (2) activated group; (3) MSCs group; (4) pre-stimulated MSCs aggregate group.

[0053] Except for the control group, anti-human CD3 antibody (1:1000) and anti-human CD28 antibody (1:2000) were added to the culture medium of the other three groups to stimulate T lymphocyte activation and proliferation. The 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO2 for 3 days, and then the proliferation of PBMC and CD4-positive T cells was detected by flow cytometry. Since the fluorescence intensity of CFSE decreases step by step with the division of cells during cell division and proliferation, the weaker the fluorescence intensity in the flow cytometer, the more proliferation generations. The ratio of umbilical cord mesenchymal stem cells to PBMC was 1:100. Except for the baseline control group, anti-human CD3 antibody (1:1000) and anti-human CD28 antibody (1:2000) were added to the culture medium of the other groups to maintain and stimulate the activation state of T lymphocytes. The 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO2 for 3 days, and then the proliferation of PBMC and CD4-positive T cells was detected by flow cytometry. Since the fluorescence intensity of CFSE decreases gradually with the division of cells during cell division and proliferation, the weaker the intensity detected by flow cytometry, the more generations of proliferation.

[0054] The results are as follows Figure 3 A and Figure 4 As shown in A, the ability of the pre-stimulated mesenchymal stem cell aggregates to inhibit the proliferation of PBMC and CD4-positive T lymphocytes was significantly improved.

[0055] Example 4: Therapeutic effect of pre-stimulated mesenchymal stem cell aggregates on the bleomycin-induced scleroderma mouse model.

[0056] The present invention proves through a systemic sclerosis model that pre-stimulated mesenchymal stem cell aggregates have a better therapeutic effect on a bleomycin-induced scleroderma mouse model.

[0057] The mouse systemic sclerosis model was established by subcutaneous injection of bleomycin (BLM). Balb / c female mice were used, aged 6-8 weeks. Sulfate-BLM was dissolved in saline to prepare a 1 mg / ml BLM solution. The back skin of the mice was shaved, and 100 μl of 1 mg / ml BLM solution was subcutaneously injected every day for 4 weeks. The specific implementation steps are as follows: 1. Twenty-four healthy Balb / c female mice were weighed and randomly divided into a healthy group (Control), a PBS group, an MSCs treatment group, and a pre-stimulated MSCs aggregate treatment group.

[0058] 2. Inject 100 μl of 1 mg / ml BLM solution subcutaneously on the back every day for 4 weeks. At the end of the second and third weeks, resuspend the prepared cells and cell aggregates in PBM buffer at a rate of 5×10 per mouse. 5 Injected into the tail vein of mice.

[0059] 3. At the end of the fourth week, the mice were killed, the skin of the lesion site was taken, rinsed with PBS, and fixed in 4% paraformaldehyde for 48 hours, followed by dehydration, embedding, sectioning, H&E staining and Masson staining to observe the histological lesions of the mouse skin. The spleen of the mouse was removed, PBS buffer (containing 2% FBS) was added, fully ground and filtered (200 mesh filter), and centrifuged at 1000g for 5 minutes at 4℃ to obtain spleen cells. 2ml of red blood cell lysis buffer was added and incubated for 5 minutes, 10ml of PBS was added to terminate the reaction, and then the supernatant was discarded by centrifugation. PBS buffer (containing 2% FBS) was added to resuspend the cells and count them.

[0060] Take 1×10 6 Spleen cells were placed in a U-bottom 96-well plate, the supernatant was discarded after centrifugation, 100 μL of cell stimulator was added, and the cells were resuspended and incubated at 37°C for 5 hours. Wash once with PBS buffer (containing 2% FBS), add 100 μL of flow cytometry antibodies CD45, TCR-β and CD4 (1:500 dilution in PBS buffer) to resuspend the cells, and incubate at 4°C in the dark for 30 minutes. After the incubation, wash once with PBS buffer containing 2% FBS, centrifuge at 1800g for 5 minutes, add 100 μL of membrane permeabilization agent and incubate at 4°C for 20 minutes, wash once with PBS buffer, centrifuge at 2000g for 5 minutes, add flow cytometry antibodies IFN-γ and IL17 (1:300 dilution in PBS buffer) respectively, mix well, incubate at 4°C in the dark for 60 minutes, wash once with PBS buffer, and detect by flow cytometry. The results are as follows Figure 5 and Figure 6 As shown, Figure 5 This is a picture of H&E staining of the skin. Figure 6 This is a Masson staining image of the skin. The collagen fibers in the skin of BLM modeled mice were significantly thickened, expanded, and tightly arranged. The dermis was significantly thickened, the blood vessels were blocked, the number of hair follicles was significantly reduced, the fat layer became thinner and disappeared, and there was inflammatory cell infiltration. In the MSCs treatment group, the degree of skin fibrosis of mice was reduced, while the degree of skin fibrosis of mice in the pre-stimulated MSCs aggregate treatment group was the smallest, and the inflammatory cell infiltration and capillary loss in the dermis were also the least. Figure 7 and Figure 8 As can be seen, a large number of Th1 and Th17 cell infiltrations were detected in the PBS group, while the pre-stimulated MSCs aggregate treatment group could inhibit the differentiation of Th1 and Th17 cells to the greatest extent.

[0061] 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 method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates, characterized in that: The following steps are involved: Umbilical cord mesenchymal stem cells are pre-stimulated with IL-6 to obtain pre-stimulated cells, which are then added to an Aggrewell 400 culture plate for centrifugation, and the centrifuged pre-stimulated cells are cultured for 12-16 hours to obtain pre-stimulated cell aggregates. The umbilical cord mesenchymal stem cells are in vitro cells.

2. The method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates according to claim 1, characterized in that: The IL-6 concentrations were 10 ng / ml, 20 ng / ml, 40 ng / ml and 80 ng / ml.

3. The method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates according to claim 1, characterized in that: The centrifugation condition of the culture plate is 1000 rpm, 5 min.

4. The method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates according to claim 1, characterized in that: The pre-stimulated cells have at least one of the following characteristics A1)-A3): A1) the expression level of immunosuppressive factors in the pre-stimulated cells is higher than that in the umbilical cord mesenchymal stem cells; A2) the ability of the pre-stimulated cells to inhibit the proliferation of human peripheral blood mononuclear cells is higher than that of the umbilical cord mesenchymal stem cells; A3) The ability of the pre-stimulated cells to inhibit the proliferation of human CD4-positive T lymphocytes is higher than that of the umbilical cord mesenchymal stem cells.

5. The method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates according to claim 1, characterized in that: The immunosuppressive factor is one or more of IDO, HGF, COX-2, and TSG6.

6. The method for preparing IL-6 pre-stimulated umbilical cord mesenchymal stem cell aggregates according to claim 1, characterized in that: The proliferation of the human peripheral blood mononuclear cells and CD4 positive T lymphocytes was detected by CFSE staining.

7. Use of pre-stimulated cell aggregates in the preparation of immunosuppressive products, characterized in that: Use of the pre-stimulated cell aggregates according to any one of claims 1 to 6 in medicine and / or vaccine.

8. Use of pre-stimulated cell aggregates in the preparation of a product for reducing inflammatory damage, characterized in that: Use of the pre-stimulated cell aggregates according to any one of claims 1 to 6 in medicine and / or vaccine.

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