Application of mesenchymal stem cells combined with methotrexate in preparation of anti-RA medicine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of mesenchymal stem cells combined with methotrexate in preparation of an anti-RA medicine. The mesenchymal stem cells are human umbilical cord mesenchymal stem cells; the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and low express CD34, CD45 and HLA-DR (Human Leukocyte Antigen-DR). The research proves that the human umbilical cord mesenchymal stem cells and methotrexate have better treatment effect compared with single human umbilical cord mesenchymal stem cells or methotrexate when being used for treating rheumatoid arthritis by combining with the human umbilical cord mesenchymal stem cells or methotrexate. RA inflammation can be effectively relieved, synovial tissue cell apoptosis can be inhibited, synovial macrophage polarization can be adjusted by regulating related signal channels, and then RA can be effectively treated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of mesenchymal stem cells combined with methotrexate in preparation of a medicine for resisting RA. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by arthritic cell infiltration, synovial inflammation, bone tissue and cartilage destruction, etc. Patients often have symmetrical joint damage or deformity, synovial inflammation, and eventually loss of joint function. At present, the specific pathogenesis of RA has not been fully and thoroughly explained. Numerous studies have shown that its pathogenesis is associated with genetic factors, external environment and immune system abnormalities. These factors are intertwined and complex, which has brought great obstacles to targeted treatment of RA.

[0003] At present, there is no specific drug and treatment plan for completely curing RA. The main treatment methods for RA are drug therapy, physical therapy and surgical treatment. Among them, drug therapy is mainly divided into glucocorticoids (GC) and non-steroidal anti-inflammatory drugs (NSAID) for inhibiting inflammation and relieving pain; disease-modifying antirheumatic drugs (DMARD), such as hydroxychloroquine, methotrexate, leflunomide and sulfasalazine, which act on the immune system pathway by targeting the control of inflammatory response. In clinical practice, anti-inflammatory analgesic drugs and immunosuppressive drugs are often used to treat the disease, and traditional Chinese medicine is also used to treat the disease. However, this treatment mode has obvious disadvantages, and the disease is prone to recurrence. In addition, long-term continuous use of drugs can also cause many adverse reactions, which adds additional pain to patients.

[0004] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are stem cells isolated and purified from umbilical cord tissue and have a wide range of applications in the field of medical treatment. The regulatory mechanisms of hUC-MSCs for various diseases are complex and have significant advantages, such as low immunogenicity, strong proliferation ability, and multiple differentiation ability, etc. hUC-MSCs can achieve therapeutic means by reducing the degree of inflammation and apoptosis in the body. Existing research reports that hUC-MSCs can be used for RA treatment. For example, CN108938670A patent reports a human umbilical cord mesenchymal stem cell preparation for treating rheumatoid arthritis, which includes mesenchymal stem cells, human blood albumin and NS. The human umbilical cord mesenchymal stem cell preparation can reduce the release of inflammatory mediators while reducing pain in patients, reduce inflammation, and thus significantly increase the body weight of patients. At the same time, it can increase the CD4+ / CD8+ ratio to varying degrees and reduce the levels of TNF-α and IL-6 in serum. For another example, CN111494421A patent reports an anti-rheumatoid arthritis human umbilical cord mesenchymal stem cell injection and a preparation method thereof. The umbilical cord mesenchymal stem cell injection includes human umbilical cord mesenchymal stem cells, human serum albumin, yam polysaccharide, complex amino acids, sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride. However, the mechanism of hUC-MSCs for treating RA is not clear, and single hUC-MSCs has limited effect on treating rheumatoid arthritis.

[0005] Therefore, it is urgent to explore new and more effective methods to break through the existing difficulties, improve the treatment effect of RA, and improve the quality of life of patients. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide the use of a pharmaceutical composition in the preparation of a medicament for treating rheumatoid arthritis. The present application researches and finds that mesenchymal stem cells combined with methotrexate have better therapeutic effect on rheumatoid arthritis than single mesenchymal stem cells or methotrexate.

[0007] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0008] The use of a pharmaceutical composition in the preparation of a medicament for treating rheumatoid arthritis, the pharmaceutical composition comprising mesenchymal stem cells and methotrexate.

[0009] As a preferred, the pharmaceutical composition consists of mesenchymal stem cells and methotrexate.

[0010] As a preferred, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0011] The present application researches and finds that human umbilical cord mesenchymal stem cells combined with methotrexate in treating rheumatoid arthritis has better therapeutic effect than single mesenchymal stem cells or methotrexate, and the two play a synergistic role in the treatment of RA.

[0012] As preferred, the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

[0013] As preferred, the human umbilical cord mesenchymal stem cells have a positive rate of CD73, CD90 and CD105 of ≥90%, and a positive rate of lowly expressed CD34, CD45 and HLA-DR of ≤5%.

[0014] As preferred, the human umbilical cord mesenchymal stem cells are extracted and separated from human umbilical cord tissues by enzyme digestion method, and are subcultured to obtain the human umbilical cord mesenchymal stem cells.

[0015] As preferred, the human umbilical cord mesenchymal stem cells are prepared by the following method:

[0016] (1) Tissue processing: obtain human umbilical cord tissue, wash and cut into small pieces of 3-5 cm in length; separate and remove the arteriovenous and intima in the umbilical cord, and retain the Wharton's jelly to cut into small pieces of 1 mm in size; add appropriate amount of collagenase to digest for 1-2 h until the tissue pieces are basically dispersed; after the cells are completely digested, filter; centrifuge the filtered cell suspension to obtain cell precipitate; 3

[0017] (4) Cell culture: mix the DMEM containing 10% FBS evenly, and resuspend the cells by gently blowing; inoculate the cells with appropriate density into a culture dish, add the same culture medium, and incubate in a culture box overnight; the next day, add the same culture medium to continue culturing the cells, and replace half of the medium after observing that the cells adhere; replace the culture medium every 5-7 days;

[0018] (5) Cell subculture: select cells with appropriate density, remove the culture medium, and wash with sterile PBS, then add trypsin to induce digestion reaction; after the cells are digested to single state, add culture medium to terminate digestion; collect the cell mixture and centrifuge to obtain cell precipitate; add fresh culture medium to the cell precipitate, and blow to form a cell suspension; the cell suspension is divided into new culture dishes, fresh culture medium is added, and the cells are continuously cultured according to the subculture ratio of 1:3.

[0019] As preferred, the rheumatoid arthritis is accompanied by joint swelling, arthritic cell infiltration, joint bone tissue destruction, joint cartilage structure destruction, synovial inflammation and / or abnormal apoptosis of synovial tissue cells.

[0020] As preferred, the treatment is achieved by the following ways:​

[0021] 1) reducing joint swelling;

[0022] and / or 2) repairing bone tissue;

[0023] and / or 3) repairing cartilage tissue;

[0024] and / or 4) relieving arthritic cell infiltration;

[0025] and / or 5) reducing the content of ACPA, IL-6 and TNF-α and increasing the content of IL-10 and TGF-β in serum;

[0026] and / or 6) inhibiting cell apoptosis: reducing the ratio of p-p65 / p65 and the ratio of Bax / Bcl-2;

[0027] and / or 7) inhibiting synovial inflammation.

[0028] As a preference, the pharmaceutical composition achieves the anti-inflammatory and anti-apoptotic effects by inhibiting the activation of the NF-κB pathway through inhibiting p65 phosphorylation and regulating the Bax / Bcl-2 signaling pathway, thereby treating RA.

[0029] The second object of the present application is to provide an application of a pharmaceutical composition in preparing a medicine for relieving inflammation of RA.

[0030] To achieve the above object, the present application adopts the following technical solution:

[0031] An application of a pharmaceutical composition in preparing a medicine for relieving inflammation of RA, wherein the pharmaceutical composition comprises mesenchymal stem cells and methotrexate.

[0032] As a preference, the pharmaceutical composition consists of mesenchymal stem cells and methotrexate.

[0033] As a preference, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0034] As a preference, the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105 and lowly express CD34, CD45 and HLA-DR.

[0035] The third object of the present application is to provide an application of a pharmaceutical composition in preparing a medicine for reducing joint swelling, repairing joint bone tissue, repairing joint cartilage tissue and / or inhibiting synovial inflammation.

[0036] To achieve the above object, the present application adopts the following technical solution:

[0037] The application relates to a pharmaceutical composition for reducing joint swelling, repairing joint bone tissue, repairing joint cartilage tissue and / or inhibiting synovial inflammation, which comprises mesenchymal stem cells and methotrexate.

[0038] Preferably, the pharmaceutical composition is composed of mesenchymal stem cells and methotrexate.

[0039] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0040] Preferably, the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

[0041] Preferably, the pharmaceutical composition promotes the transformation of macrophages in the synovium from M1 type promoting inflammation to M2 type inhibiting inflammation.

[0042] Preferably, the medicine inhibits the expression of pro-inflammatory factors, promotes the expression of anti-inflammatory factors, and improves the CD163 / CD86 ratio; the pro-inflammatory factors include IL-6; and the anti-inflammatory factors include IL-10.

[0043] The fourth object of the application is to provide a pharmaceutical composition for treating rheumatoid arthritis.

[0044] To achieve the above object, the application adopts the following technical scheme:

[0045] The pharmaceutical composition for treating rheumatoid arthritis comprises mesenchymal stem cells and methotrexate.

[0046] Preferably, the pharmaceutical composition is composed of mesenchymal stem cells and methotrexate.

[0047] Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0048] Preferably, the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

[0049] Preferably, the pharmaceutical composition is a combination drug; or the pharmaceutical composition is prepared into any dosage form of a pharmaceutically acceptable compound medicine.

[0050] The application has the following beneficial effects:

[0051] 1. Currently, there is no report on the synergistic effect of hUC-MSCs combined with methotrexate in the treatment of rheumatoid arthritis. The present application proves that the effect of hUC-MSCs combined with methotrexate in the treatment of rheumatoid arthritis is obviously better than that of hUC-MSCs or MTX alone. Both have synergistic effect in the treatment of RA, and play a role in anti-inflammatory and anti-apoptosis, and effectively reduce inflammatory response, including reducing inflammatory cell infiltration, repairing joint bone and cartilage tissue, reducing the content of pro-inflammatory factors in serum, and increasing the content of anti-inflammatory factors, and thus relieving the disease. The present application provides a new and reliable scientific theoretical basis for the clinical treatment of rheumatoid arthritis.

[0052] 2. This research focuses on hUC-MSCs and the combination of hUC-MSCs and methotrexate for the first time, and deeply explores the internal mechanism of treating RA by means of NF-κB signaling pathway and Bax / Bcl-2 apoptosis pathway. The present application proves that hUC-MSCs and hUC-MSCs combined with methotrexate can inhibit the activation of NF-κB signaling pathway, and inhibit the Bax / Bcl-2 apoptosis pathway. hUC-MSCs combined with methotrexate can play a role in anti-inflammatory and anti-apoptosis, and realize the effective treatment of RA through the synergistic effect of multiple aspects. This discovery reveals the potential molecular mechanism of hUC-MSCs and methotrexate in treating RA, and provides a new theoretical basis and treatment idea for the clinical treatment of RA, which is expected to promote the further optimization and innovation of RA treatment method.

[0053] 3. The present application proves that single hUC-MSCs and hUC-MSCs combined with methotrexate can promote the transformation of synovial macrophages from M1 type to M2 type, thereby reducing the level of inflammation (reducing the level of pro-inflammatory factors and increasing the level of anti-inflammatory factors), and the combined treatment of hUC-MSCs and MTX is the most significant, which proves that hUC-MSCs+MTX combined drug is a potential candidate drug for anti-RA, and provides a new treatment method for RA treatment. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Figure for subculture of hUC-MSCs;

[0055] Figure 2 Figure for flow detection of hUC-MSCs surface markers, wherein, Figure 2 A is the flow detection result figure of CD73; Figure 2 B is the flow detection result figure of CD90; Figure 2 C is the flow detection result figure of CD105; Figure 2 D is the flow detection result figure of CD34; Figure 2 E is the flow detection result figure of CD45; Figure 2 -F is the flow cytometry test result of HLA-DR;

[0056] Figure 3 This is the result of hUC-MSCs differentiation ability identification, among which, Figure 3 -A is a diagram of adipogenic differentiation of hUC-MSCs; Figure 3 -B is a diagram of osteogenic differentiation; Figure 3 -C is a diagram of chondrogenic differentiation;

[0057] Figure 4 Figure 2 shows the weight and joint scores of rats, where Figure 4 -A is the graph showing the changes in rat body weight; Figure 4 -B is the result of rat arthritis scoring;

[0058] Figure 5 Figure 2 shows the serum CCP levels of rats in each group on days 10 and 20, ****p < 0.001;

[0059] Figure 6 Graph showing the body weight of rats in each group; n = 7, *P ≤ 0.05, **P ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001;

[0060] Figure 7 This is the rat joint scoring diagram, where Figure 7 -A is a graph showing the swelling of the joints of rats in each group before and after treatment; Figure 7 -B is the arthritis score results of rats in each group before and after treatment; compared with the CIA group, *P≤0.05, **P≤0.01, ***p≤0.001, ****p≤0.0001;

[0061] Figure 8 This is the result of EdU detection of hUC-MSCs colonization in rat joints after 3 days;

[0062] Figure 9 This is the result of EdU detection of hUC-MSCs colonization in rat joints after 7 days;

[0063] Figure 10 Figure 2 is the HE staining and analysis of the knee joints of rats in each group, among which, Figure 10 -A is the HE staining image of the knee joints of rats in each group; Figure 11 This is the knee joint tissue injury score chart; compared with the CIA group, *P≤0.05, **P≤0.01, ***P≤0.001;

[0064] Figure 11 The Masson staining and analysis of the joints of rats in each group are shown in Figure 2. Figure 11-A is the Masson staining chart of the joints of the Control group rats; Figure 11 -B is the Masson staining chart of the joints of the CIA group rats; Figure 11 -C is the Masson staining chart of the joints of the CIA+MSC group rats; Figure 11 -D is the Masson staining chart of the joints of the CIA+MTX group rats; Figure 11 -E is the Masson staining chart of the joints of the CIA+MSC+MTX group rats; Figure 11 -F is the Masson positive rate statistical chart of the rats in each group; compared with the CIA group, *P≤0.05, **P≤0.01, ***P≤0.001;

[0065] Figure 12 A chart of the Safranin O-fast green staining and analysis of the joints of the rats in each group, wherein, Figure 12 -A is the Safranin O-fast green staining chart of the joints of the Control group rats; Figure 12 -B is the Safranin O-fast green staining chart of the joints of the CIA group rats; Figure 12 -C is the Safranin O-fast green staining chart of the joints of the CIA+MSC group rats; Figure 12 -D is the Safranin O-fast green staining chart of the joints of the CIA+MTX group rats; Figure 12 -E is the Safranin O-fast green staining chart of the joints of the CIA+MSC+MTX group rats; Figure 11 -F is the Safranin O-fast green staining positive rate statistical chart; compared with the CIA group, *P≤0.05, **P≤0.01;

[0066] Figure 13 A chart of the determination results of the cytokine content in serum, wherein, Figure 13 -A is a chart of the CCP content change of the rats before and after treatment; Figure 13 -B is a chart of the pro-inflammatory factor IL-6 content change of the rats before and after treatment; Figure 13 -C is a chart of the pro-inflammatory factor TNF-α content change of the rats before and after treatment; Figure 13 -D is a chart of the anti-inflammatory factor IL-10 content change of the rats in each group before and after treatment; Figure 13 -E is a chart of the anti-inflammatory factor TGF-β content change of the rats in each group before and after treatment;

[0067] Figure 14 A chart of the protein expression amount of inflammatory factors;

[0068] Figure 15 A chart of the Western blot expression analysis of the macrophage marker in synovial tissue, wherein, Figure 15 -A is a chart of the Western blot detection results of the expression levels of CD86 and iNOS; Figure 15-B is the graph of the analysis of the expression of CD86 and iNOS in each group of rats; Figure 15 -C is the graph of the results of the Western blot for the expression of CD163; Figure 15 -D is the graph of the analysis of the expression of CD163 in each group of rats; all experiments were performed in triplicate and the Student's t test was used to analyze the significance of the experiments; error: *P < 0.05; **P < 0.01; ***p < 0.001; ns is not statistically significant;

[0069] Figure 16 is the graph of the results of the qRT-PCR expression analysis of the macrophage markers in the synovial tissue, wherein, Figure 16 -A is the graph of the expression of CD86, iNOS and IL-6 in the synovial tissue; Figure 16 -B is the graph of the expression of CD163 and IL-10 in the synovial tissue; Figure 16 -C is the graph of the ratio of the expression of CD163 / CD86; each group was performed in at least triplicate and the results were analyzed for significance based on the Student's t test. *P < 0.05, **p < 0.01, ***p < 0.001;

[0070] Figure 17 is the graph of the immunohistochemical staining of the synovial tissue, wherein, Figure 17 -A is the graph of the F4 / 80 immunohistochemical staining of the synovial tissue of the rats of the Control group; Figure 17 -B is the graph of the F4 / 80 immunohistochemical staining of the synovial tissue of the rats of the CIA group; Figure 17 -C is the graph of the F4 / 80 immunohistochemical staining of the synovial tissue of the rats of the CIA + MSC group; Figure 17 -D is the graph of the F4 / 80 immunohistochemical staining of the synovial tissue of the rats of the CIA + MTX group; Figure 17 -E is the graph of the F4 / 80 immunohistochemical staining of the synovial tissue of the rats of the CIA + MSC + MTX group; Figure 17 -F is the graph of the quantification of the results of the immunohistochemical staining of the rats of each group;

[0071] Figure 18 is the graph of the results of the immunohistochemical staining of the synovial tissue, wherein, Figure 18 -A is the graph of the results of the immunohistochemical staining of CD86 and CD163 in the synovial tissue of the rats of each group; Figure 18 -B is the graph of the quantification of the results of the immunohistochemical staining of CD86 in the synovial tissue of the rats of each group; Figure 18 -C is the graph of the quantification of the results of the immunohistochemical staining of CD163 in the synovial tissue of the rats of each group;

[0072] Figure 19 is the graph of the expression of the proteins of the NF-κΒ signaling pathway and of the Bax / Bcl-2 signaling pathway;

[0073] Figure 20 The figure shows the apoptosis of synovial tissue cells detected by TUNEL method; Figure 20 -A is the result of TUNEL assay to detect cell apoptosis in synovial tissue of rats in each group; Figure 20 -B is for Figure 20 -A is a graph quantifying the experimental results; compared with the CIA group, n=7, *P<0.05; **P<0.01, ***P<0.001, ****P<0.001. DETAILED DESCRIPTION

[0074] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0075] This study used hUC-MSCs and an RA rat model as research subjects, exploring the effects of hUC-MSCs on RA inflammation, apoptosis, and synovial macrophage polarization through in vivo animal experiments. The study also explored whether hUC-MSCs could be combined with methotrexate (MTX), a traditional RA treatment, to achieve synergistic therapeutic effects, and explored the mechanism of action of hUC-MSCs in anti-RA signaling pathways.

[0076] The main research contents and results of the present invention are as follows:

[0077] 1. Extracting hUC-MSCs with self-renewal and multiple differentiation capabilities from human umbilical cord tissue

[0078] Human umbilical cord tissue was extracted and purified using an enzymatic digestion method and then subcultured. To investigate the characteristics of the extracted cells, cell morphology was analyzed under a microscope, stem cell surface markers were identified using flow cytometry, and the stem cells were induced to become adipogenic, osteogenic, and chondrogenic under different conditions, and the induced differentiated cells were identified. The results showed that the hUC-MSCs extracted using the enzymatic digestion method had excellent self-renewal, high proliferation capacity, and multidirectional differentiation ability, and the cell surface contained stem cell-specific markers. Under different induction conditions, the stem cells demonstrated the potential to differentiate into various tissues, including adipose tissue, bone tissue, and cartilage tissue.

[0079] 2. Through effective means, a rat disease model of RA was successfully constructed.

[0080] The present application adopts bovine collagen type II (CII) and complete Freund's adjuvant (CFA) to establish a RA rat model (CIA). Whether the modeling is successful is judged through various identifications. On the one hand, the joints of the rats are scored in detail, and the appearance, swelling degree, activity and the like of the joints are quantitatively evaluated; on the other hand, the self-antibody and inflammatory factor and the like in the serum of the rats are detected and analyzed. The results show that after the SD rats are immunized for multiple times by using CII+CFA emulsion, the arthritis score, the RA self-antibody and the inflammatory factor in the blood rise and the like. The model has high similarity with the human RA disease in pathological performance, and can accurately simulate the pathological process of the human RA disease. Comprehensive comments show that the bovine CII and CFA successfully induce the CIA rat model. The successfully established animal model provides reliable and effective test animal model basis for in-depth research on the cause and pathogenesis of RA and exploration of new treatment methods, and is helpful for researchers to further carry out related research work and promote the development of the RA treatment field.

[0081] 3. hUC-MSCs have certain therapeutic effect on CIA rats

[0082] In order to explore the therapeutic effect of hUC-MSCs, the present application adopts tail vein injection of hUC-MSCs, MTX gavage and combined use of hUC-MSCs and MTX to treat the CIA rats, arthritis scores of the rats in each group are obtained, HE staining is adopted to explore the inflammatory cell infiltration of arthritis, Masson staining is adopted to detect the repair ability of bone tissue and ponceau O fast green staining is adopted to detect the repair ability of cartilage, Elisa is adopted to detect the change of inflammatory factors in the serum of each group, and a series of explorations are carried out on the protein expression of inflammatory factors of the rats in each group through Western blot. The research results show that various treatment methods have certain therapeutic effect on the CIA rats: the inflammatory cell infiltration of the CIA rats in each treatment group is reduced, the joint bone tissue is restored and the cartilage is repaired, the pro-inflammatory factors in the serum are reduced, the anti-inflammatory factors are increased, and the protein expression of inflammatory factors is reduced. Further comparison shows that the therapeutic effect of the combined treatment of hUC-MSCs and MTX is obviously better than that of hUC-MSCs or MTX alone, and the inflammatory indexes of the combined treatment are lower. This result powerfully proves that in the in-vivo environment, hUC-MSCs and MTX have synergistic effect in treating RA, and the two can cooperate with each other to jointly play the anti-inflammatory role, more effectively reduce the inflammatory reaction and relieve the disease. The present application provides a new and reliable scientific theoretical basis for the clinical treatment of rheumatoid arthritis, and is expected to provide new treatment ideas and scheme selection for the clinicians in treating RA, promote the development and progress of the clinical treatment of RA, and bring more treatment hopes for the RA patients.

[0083] 4. hUC-MSCs promote the transformation of synovial macrophages from M1 to M2

[0084] In order to further study the effect on macrophage polarization in synovial tissue, the present invention detected markers of various phenotypes of macrophages in the synovial tissue of each group of rats by qRT-PCR, Western blot, immunohistochemistry and other methods. The results showed that the synovial tissue macrophages in each treatment group had a trend of transformation from the pro-inflammatory M1 type to the anti-inflammatory M2 type. All treatment groups showed the effect of reducing the level of inflammation, and the overall level of pro-inflammatory factors decreased, while the level of anti-inflammatory factors increased. Among them, the combined treatment effect of hUC-MSCs and MTX was the most significant, proving that the combination of hUC-MSCs + MTX is a potential anti-RA drug candidate and provides a new treatment method for RA treatment.

[0085] 5. hUC-MSCs play an anti-RA role by regulating the expression of NF-κB signaling pathway and apoptosis pathway.

[0086] To gain deeper insights into the pathogenesis of RA, this study conducted a detailed investigation of the inflammatory pathways involved in its pathogenesis. The NF-κB and apoptosis signaling pathways are currently believed to play a crucial role in RA pathogenesis. Upon activation, they regulate the expression and transcription of several key proinflammatory cytokines. We hypothesized that hUC-MSCs exert their anti-RA effects through NF-κB signaling and anti-apoptosis. We performed ELISA assays for relevant cytokines in the serum of rats in each group, and used Western blot and qRT-PCR to investigate the anti-RA effects and mechanisms of hUC-MSCs and MTX. The results showed that hUC-MSCs and MTX modulated the expression of the NF-κB signaling pathway during RA treatment. Specifically, both hUC-MSCs and MTX exerted their anti-inflammatory effects by downregulating the expression of proteins involved in the inflammatory pathway, including p-p65 and the apoptosis-related protein Bax. Furthermore, both hUC-MSCs and MTX exhibited anti-apoptotic effects, including upregulation of the anti-apoptotic protein Bcl-2, which in turn inhibited the apoptotic process of synovial cells. In summary, the combination of hUC-MSCs and MTX has a potent anti-RA effect, modulating relevant signaling pathways and achieving a favorable therapeutic effect. This discovery reveals the potential molecular mechanism of hUC-MSCs and MTX in treating RA, providing a new theoretical basis and therapeutic approach for the clinical treatment of RA, and is expected to promote further optimization and innovation in RA treatment methods.

[0087] In the embodiment of the present invention, the key Chinese and English abbreviation information is detailed in Table 1.

[0088] Table 1 Chinese and English abbreviations

[0089]

[0090]

[0091] Example 1. Isolation, culture and identification of hUC-MSCs

[0092] MSCs were isolated and extracted from human umbilical cord tissue and subcultured. Their morphological characteristics were observed and their surface markers and multidirectional differentiation potential were identified. The specific methods and results are as follows:

[0093] 1. Experimental Materials

[0094] (1) Tissue samples

[0095] The human umbilical cord mesenchymal stem cells (hUC-MSCs) used in this study were extracted from umbilical cord tissue provided by Chongqing Ninth People's Hospital and passed the review of the Medical Ethics Committee of Chongqing Ninth People's Hospital (No.: 2024-科w(伦申)-001) and were cultured and passaged in this laboratory.

[0096] (2) Preparation of main reagents

[0097] 1× PBS (0.01M Phosphate Buffered Saline): Take 1L of PBS (phosphate buffered saline) and mix thoroughly with 1L of distilled water. Once completely dissolved, the resulting solution has a concentration of 0.01M and a pH of 7.2-7.4. Autoclave and store in a cool, dark place for long-term storage.

[0098] 10% serum conventional culture medium: Mix 500 ml of culture medium and 50 ml of serum, and use for culturing cells.

[0099] Cell Cryopreservation Solution: First, measure 9 volumes of serum into a 50ml centrifuge tube. Then, add 1 volume of DMSO and mix thoroughly to obtain a cell cryopreservation solution that meets the requirements. Once the cell cryopreservation solution is prepared, it can be used in subsequent cryopreservation experiments to effectively ensure the long-term storage of cells.

[0100] 2. Experimental Methods

[0101] 1. Cell culture

[0102] (1) Tissue processing

[0103] 1) After obtaining the umbilical cord tissue, carefully rinse the surface of the tissue with saline until all blood is removed. Then, using appropriate tools, cut the cleaned umbilical cord tissue into small segments of 3-5 cm in length for subsequent processing and research.

[0104] 2) In a clean bench, use forceps and scissors to carefully separate and remove the arteries, veins and endothelium of the umbilical cord, retain Wharton's jelly and cut it into pieces of about 1 mm 3 Prepare small pieces of the same size for subsequent experimental steps.

[0105] 3) Add an appropriate amount of collagenase and digest for 1-2 hours until the tissue blocks are basically discrete. When the cells are completely digested, filter them using a filter. The purpose of this step is to effectively remove undigested tissue debris and ensure the purity of the cell suspension. Subsequently, the filtered cell suspension is centrifuged. Under the action of centrifugal force, the cells are precipitated to the bottom of the centrifuge tube, thereby obtaining the required cell pellet for subsequent experimental operations.

[0106] (2) Cell culture

[0107] 1) Mix well with DMEM containing 10% FBS and gently pipette to resuspend the cells. Seed the cells at an appropriate density in a culture dish, add the same culture medium, and continue culturing in an incubator.

[0108] 2) On the second day, add 10 mL of the same culture medium and continue culturing the cells. After observing that the cells have attached, change half of the medium to avoid excessive accumulation of metabolic waste and adverse effects on the cells. Change the medium every 5-7 days.

[0109] (3) Cell passage

[0110] 1) First, select cells that meet the required density. Remove the culture medium from the cell culture vessel and gently wash the cells with sterile PBS to remove any remaining culture medium and impurities. After washing, add an appropriate amount of trypsin to the culture vessel to promote cell digestion.

[0111] 2) When the cells have been digested and separated into single cells, add an appropriate amount of culture medium to terminate the digestion. The cell mixture is then collected and transferred to a centrifuge tube. Centrifuge to pellet the cells.

[0112] 3) After centrifugation, discard the supernatant. Add fresh culture medium to the centrifuge tube containing the cell pellet and gently pipette the cells to form a cell suspension.

[0113] 4) Based on the specific needs of subsequent experiments, evenly distribute the prepared cell suspension into new culture dishes. Add an appropriate amount of fresh culture medium to each dish and continue culturing the cells at a 1:3 cell culture ratio. Place the dish in a suitable culture environment to ensure normal cell growth and proliferation.

[0114] (4) Cell cryopreservation

[0115] 1) Take the third generation P3 cells, observe their growth state and density under a microscope, and remove the culture medium when the cell density reaches about 80%. Then wash the cells with sterile PBS, and add an appropriate amount of trypsin to digest the cells.

[0116] 2) After digestion into single cells, stop the digestion by adding an appropriate amount of culture medium, and collect and centrifuge them.

[0117] 3) Prepare the cell freezing solution in advance, then add the freezing solution to the cells in a 1:1 ratio for resuspension. Carefully transfer the obtained cell suspension to a freezing tube. Then, clearly label the specific time, the name of the operator, and the name of the frozen cells on the freezing tube. After labeling, perform gradient cooling on the cells, and then quickly place the freezing tube in a liquid nitrogen tank for long-term frozen storage of the cells.

[0118] (5) Cell recovery

[0119] Turn on the ultraclean bench and ultraviolet light for sterilization in advance, and turn on the constant temperature water bath and set the temperature to 37°C. Take the frozen cells out of the liquid nitrogen tank and isolate them with PE gloves, and place them in the water bath for 3-5 min for thawing. After thawing, place them in the centrifuge and set the speed to 1000 rpm for 5 min of centrifugation. Slowly pour off the supernatant, add an appropriate amount of culture medium to resuspend the cells, and inoculate them in a culture dish, and continue to culture them in a 37°C, 5% CO2 incubator.

[0120] 2. Identification of cell surface markers

[0121] Take the third generation cells, discard the culture medium, wash them with PBS, trypsinize the cells, add a single cell suspension, and inoculate them in a twelve-well plate. After 5 days of inoculation, detect the cell density and observe the proliferation according to 1 x 10 6 / 5 μl 100 μl of the corresponding fluorescent antibody dye (anti-CD90-APC, anti-CD73-APC, HLA-DR, anti-CD105-PE, anti-CD45-PE, anti-CD34-PerCP) is added. Incubate on ice for 20 min in the dark, wash with cell staining buffer, centrifuge at 350 x g for 5 min, resuspend with cell buffer, and detect on a flow cytometer. Analyze with FlowJo software.

[0122] 3. Identification of hUC-MSCs differentiation ability

[0123] (1) Osteogenic differentiation induction and identification

[0124] 1) Take a six-well plate, add 1 mL of gelatin with a concentration of 0.1% to each well, shake gently to make the gelatin evenly distributed and fully cover the bottom of the well. Then, place the six-well plate in the incubator on the clean bench and let it stand for not less than 30 min. After standing, remove the gelatin in the well.

[0125] 2) Seed at a density of 2 x 10 4 cells per square centimeter, add 2 mL of regular complete medium to each well, and then place the seeded six-well plate in a carbon dioxide incubator with a temperature of 37°C, a carbon dioxide content of 5%, and a humidity saturation, and culture the cells.

[0126] 3) During cell culture and induction differentiation experiments, closely observe the growth state of the cells. When the degree of cell fusion is observed to be exactly 70%, carefully aspirate the original complete medium in the six-well plate well with a pipette, and add 2 mL of OriCell human related stem cell osteogenic induction differentiation medium to each well. In the subsequent culture stage, replace the fresh osteogenic induction differentiation medium for the cells once every 3 days to maintain the nutritional environment and signal stimulation required for cell induction differentiation. At the same time, continuously observe the morphological changes and growth trend of the cells, and induce culture for 2-4 weeks according to the actual situation.

[0127] 4) After the induction culture stage is completed, enter the staining identification link. First, aspirate the osteogenic induction differentiation complete medium in the six-well plate, and try to be gentle during the operation to prevent the cells from being blown away. Then, slowly add an appropriate amount of PBS buffer to each well to wash the cells 2-3 times. After washing is completed, add 2 mL of 4% paraformaldehyde solution to each well, and fix for 30 min at room temperature. After fixation is completed, carefully aspirate the fixing solution, and wash the cells with PBS buffer again 2-3 times to completely remove the residual fixing solution. Next, accurately add 2 mL of alizarin red staining solution to each well, and stain at room temperature for 5-10 min to fully stain the mineralized nodules of osteoblasts. After staining is completed, aspirate the staining solution in time, and wash the cells with PBS buffer repeatedly 2-3 times to ensure that the excess staining solution is completely removed to avoid affecting the subsequent observation effect. Finally, inject 2 mL of PBS buffer into each well, and place the culture plate stably under a microscope to carefully observe the osteogenic staining effect of the cells.

[0128] (2) Adipogenic differentiation induction and identification

[0129] 1) Cell treatment is the same as above.

[0130] 2) When the cell fusion reaches 100%, aspirate the complete medium in each well, and add 2 mL of OriCell human related stem cell adipogenic induction differentiation medium A to each well.

[0131] 3) After 3 days of induction, aspirate the A solution from the six-well plate, and then add 2 mL of OriCell human related stem cell adipogenic induction medium B solution to each well to provide a new induction environment for the cells and promote further differentiation of the cells in the adipogenic direction.

[0132] 4) After 1 day of induction in the B solution, replace the B solution with the A solution to continue the induction process. In this way, the A solution and the B solution are alternately used, and the state of the cells is observed during this period, focusing on the generation of lipid droplets.

[0133] 5) When the adipogenic induction and differentiation stage is complete, aspirate the complete medium, and wash the cells 2-3 times with PBS buffer to remove residual medium components, preparing for subsequent fixation and staining operations.

[0134] 6) Cell fixation step: after washing is complete, add 2 mL of 4% paraformaldehyde solution to each well, and let it stand for 10 minutes at room temperature for fixation. Then wash twice with PBS, add an appropriate amount of staining washing solution to evenly cover the cells, and let it stand for 20 seconds.

[0135] 7) Staining and post-processing: after aspirating the staining washing solution, add an appropriate amount of modified oil red O staining agent, and perform 10-20 minutes of staining. After staining is complete, aspirate the oil red O staining solution, add an appropriate amount of staining washing solution again, and maintain for 30 seconds to preliminarily remove excess staining agent. Then continue to wash repeatedly and thoroughly with PBS buffer until the staining solution is completely washed away to avoid interference with subsequent observation by residual staining agent.

[0136] 8) Observe the adipogenic effect under a microscope.

[0137] (3) Chondrogenic differentiation induction and identification

[0138] 1) Take 3-4 x 10 5 hUC-MSCs and carefully transfer them to a 15 mL centrifuge tube. Place the centrifuge tube in a 20°C environment, set the centrifuge parameters to 250 x g, and run for 4 minutes. After centrifugation is complete, aspirate the supernatant, and leave the cell pellet at the bottom of the tube.

[0139] 2) Add 0.5 mL of chondrogenic induction and differentiation premix to the centrifuge tube containing the cell pellet, and gently blow to resuspend the pellet. Then place the centrifuge tube again in a 20°C environment, and centrifuge at 150 x g for 5 minutes to promote contact between the cells and the premix and preliminary aggregation.

[0140] 3) Repeat the above step twice for washing.

[0141] 4) Take 10 μL of OriCel human related stem cell chondrogenic induction and differentiation additive II and mix well based on 1 mL of chondrogenic induction and differentiation premix.

[0142] 5) Cell Seeding: Gently resuspend the cell pellet in 0.5 mL of freshly prepared complete chondrogenic differentiation medium and centrifuge again at 150 × g, 20°C for 5 min. After centrifugation, loosen the cap of the centrifuge tube to ensure smooth gas exchange. The centrifuge tube is then placed upright in an incubator set at 37°C, 5% CO2, and saturated humidity to allow chondrogenic differentiation.

[0143] 6) Change the cell culture medium every 2-3 days by adding approximately 0.5 mL of freshly prepared complete chondrogenic differentiation medium to each tube. After each change, gently tap the tube to gently shake the chondrocytes loose from the bottom of the tube and suspend them in the culture medium, maintaining a continuous and stable state of induced differentiation.

[0144] 7) When cartilage balls with a diameter of 1.5-2 mm are formed, the relevant samples are processed according to the following process for subsequent section staining.

[0145] Fixation: Wash the chondrocytes carefully with 1×PBS and immerse them in 4% paraformaldehyde solution for more than 30 minutes.

[0146] Dehydration: Place the samples in 50%, 70%, 80%, 95% alcohol solutions and anhydrous alcohol in sequence. Each concentration of alcohol treatment lasts for 30 minutes to ensure the adequacy and stability of the dehydration process and prepare for subsequent experimental steps.

[0147] Transparent: ① Mix xylene and anhydrous alcohol in a ratio of 1:1 and soak the cartilage balls for 2 hours; ② Soak in pure xylene for 1.5 hours; ③ Soak in fresh xylene for 1 hour;

[0148] Wax immersion: ① Mix xylene and paraffin wax in a ratio of 1:1 and immerse the chondrocytes in the mixture in a 40°C oven for 40 minutes; ② Soak the chondrocytes in pure paraffin wax in a 55°C oven for 30 minutes;

[0149] Embedding: Take out the cartilage ball and place it in the mold, pour in paraffin, let it stand and cool, and trim the wax block after cooling;

[0150] Sectioning: Cut the wax block and make serial sections, each section is 3-7 μm thick;

[0151] Sticking: Place the sliced ​​cartilage balls on a clean glass slide. After placement, move the slide into an oven set at 35°C to dry the slices at this temperature.

[0152] Dewaxing: ① First, immerse the sample in pure xylene for 15 minutes to fully dissolve the paraffin wax. After 15 minutes, carefully pour out the used xylene, replace it with fresh xylene, and immerse the sample again for 10 minutes to further ensure effective dewaxing. ② Use a 1:1 ratio of xylene to absolute alcohol for 10 minutes. ③ After completing the previous steps, immerse the sample in 95%, 85%, 70%, and 50% alcohol solutions for 10 minutes each. After immersion, rinse the sample with tap water to remove any residual alcohol for subsequent experimental procedures.

[0153] 8) Safranin O-Fast Green Staining

[0154] ① Stain with hematoxylin for 5-10 minutes, rinse with tap water to remove excess stain, then place in a hydrochloric acid-alcohol solution (1% hydrochloric acid, 70% ethanol) for 30 seconds. Rinse in tap water for 10 minutes to return the cartilage to blue. ② Stain with Fast Green for 5 minutes. Rinse rapidly in tap water to remove excess stain until the cartilage is colorless. ③ Differentiate in acetic acid differentiation solution for 30 seconds, then rinse in tap water for 10-30 seconds. ④ Stain with Safranin O for 8 minutes. ⑤ Dehydrate rapidly three times with anhydrous ethanol.

[0155] 9) Transparency and sealing: Transparency with xylene for 3 × 5 min, and sealing with neutral resin.

[0156] 10) Observe the cells under a microscope, record their morphological characteristics, and pay attention to the staining of the cells.

[0157] 3. Experimental Results

[0158] (1) Morphological observation

[0159] hUC-MSCs were isolated from human umbilical cord tissue using an enzymatic digestion method. After 3-5 days of culture, adherent cells grew rapidly, reaching approximately 90% confluence. Most cells exhibited fibroblast-like growth, while a few showed spindle-shaped or irregular triangles. The cells were uniform in size. After passage, the cells grew rapidly, reaching 80%-90% confluence within a week. For details, see Figure 1 .

[0160] (2) Surface marker identification

[0161] Flow cytometry was used to detect hUC-MSCs surface markers. The results showed that hUC-MSCs specific surface antigens CD73, CD90, and CD105 were strongly positive, while CD34, CD45, and HLA-DR were negative. Figure 2 .

[0162] (3) Identification of differentiation ability

[0163] hUC-MSCs were induced to differentiate into adipocytes for about 2 weeks. Under an inverted microscope, the cell morphology changed from long and thin to large and round, and clear oil droplet-like substances appeared in the cytoplasm. After oil red O staining, the cells were red, as shown in Fig. 4A. Figure 3 When hUC-MSCs were induced to differentiate into osteoblasts for about 3 weeks, under an inverted microscope, the cell morphology was irregular polygonal, and opaque areas were visible. After alizarin red staining, dense red precipitates were observed in the form of nodules, as shown in Fig. 4B. Figure 3 When hUC-MSCs were induced to differentiate into chondrocytes for about 2 weeks, under an inverted microscope, the cells aggregated into small spheres with a diameter of about 1 mm. After Safranin O-fast green staining, the cells were light green, as shown in Fig. 4C. Figure 3 These results indicate that hUC-MSCs have good differentiation capacity into adipocytes, osteoblasts, and chondrocytes.

[0164] Four, summary and discussion

[0165] In this example, hUC-MSCs were isolated from human umbilical cord tissue by enzyme digestion and subcultured. The cells were identified by morphology and cell surface markers, and their osteogenic, adipogenic, and chondrogenic differentiation was studied in vitro. The cells grew in a fibroblast-like manner, with most cells adhering to the substrate and having strong adhesion ability. Flow cytometry was used to identify the surface markers of the cells, and the results showed that the positive rates of CD73, CD90, and CD105 were ≥90%. The expression of CD34, CD45, and HLA-DR was also investigated, and the results showed that the positive rates were not more than 5%. After completing the induction and differentiation program, the cells were stained with alizarin red, oil red O, and Safranin O-fast green, and the staining results were observed and analyzed under a microscope. The results showed that the cells exhibited osteogenic, adipogenic, and chondrogenic differentiation tendencies. The alizarin red-positive reaction was observed in the osteogenic area, the oil red O staining produced a specific color in the adipogenic area, and the Safranin O-fast green staining also had a distinct feature in the chondrogenic area, which fully confirmed that the cells had good and multiple differentiation potential. The results indicated that the umbilical cord tissue was rich in hUC-MSCs with high proliferation and strong differentiation capacity, and this study laid a foundation for the research and application of hUC-MSCs.

[0166] Example 2. Establishment of a collagen-induced RA rat model

[0167] In this example, a CIA model was established in rats using bovine collagen type II (CII) and complete Freund's adjuvant (CFA). The morphology and serology of the animal disease model were verified and explored.

[0168] I. Experimental materials

[0169] (1) Rats

[0170] Healthy male SD rats (SPF level), 35, body weight 200-220 g, 8-9 weeks old. The experimental animals were provided by Jingda Experimental Animal Co., Ltd. The experiment was approved by the Ethics Committee of Southwest University to ensure that the experiment was conducted in accordance with ethical standards.

[0171] (2) Modeling reagent preparation

[0172] According to the volume 1:1, take appropriate amount of CII and CFA to prepare a solution with a mass concentration of 1 g / L, that is, 1 mg of CII per 1 mL of solvent. The specific steps include: to prevent collagen denaturation, the whole process is carried out on ice. Use 2 1 ml sterile syringes to mix collagen and adjuvant by connecting a three-way valve, repeatedly pumping back and forth with the syringe until the solvent appears milky white, and when the emulsion is dropped into water, the sample forms a tight droplet in water and does not spread out. When it spreads out, it should be re-mixed until a stable emulsion is formed.

[0173] II. Experimental method

[0174] 1. Grouping of SD rats

[0175] After weighing each healthy male SD rat accurately, arrange them in order from small to large (or from large to small) according to the obtained weight values. Then, use the random number function in EXCEL software to generate random numbers, and randomly divide the 35 rats into 2 different groups according to the results of the random numbers: normal saline control group (n=7); CII+CFA model group (n=28), housed in separate cages, 3-4 rats per cage.

[0176] 2. Model establishment

[0177] Weigh each group of rats, disinfect the tails of the rats in the modeling group with 75% alcohol, and inject the emulsion subcutaneously according to 200 μL per SD rat. Choose the skin about 1.5 cm from the tail root to avoid rat blood vessels, and inject multiple small doses multiple times. When a stable round small papule is seen on the skin surface after injection, it is considered successful, and the needle is slowly pulled out. The time of the first immunization is set as 0d, which is used as the starting point for calculation. On the 7th day after the first immunization, the same operation procedure and method as the first immunization are used to immunize each rat twice, and the injection dose of each rat is fixed at 100 μL of immune emulsion.

[0178] 3. CIA model verification

[0179] (1) Observation of rat body weight and clinical manifestations

[0180] The rats were weighed, hair luster, joint paw redness, joint lesions, etc. every 3 days from the first immunization. The joint conditions of each rat were observed and evaluated in detail according to the pre-prepared arthritis index scoring standard shown in Table 2, and the four limbs were accurately scored according to the indicators and scores specified in the scoring standard, so as to obtain quantitative data reflecting the degree of arthritis in rats.

[0181] Table 2. Arthritis index scoring standard according to 0-4 grade scoring

[0182] Score Joint manifestation characteristics 0 No signs of erythema or swelling 1 Erythema and mild swelling limited to the tarsal or ankle joints 2 Erythema and moderate swelling extending from the ankle to the metatarsal joints 3 Erythema and severe swelling extending from the metatarsal joints to the toes 4 Erythema and severe swelling involving the ankles, feet, and toes; or stiffness in the limbs

[0183] The success criteria of CIA modeling are as follows:

[0184] After modeling, the body weight slightly decreased, the hair luster decreased, the spirit was depressed, the food and activity amount decreased compared with before; 24h after immunization, the model group rats had red and thick tail, often accompanied by fever, and the foot sole began to have redness. After the second immunization, the redness of the foot sole of the rats continued to intensify, and the arthritis symptoms became more significant. At the same time, some rats also had inflammatory nodules on the tail, which further indicated the spread and spread of inflammation in the body of the rats; the arthritis index score was greater than or equal to 4.

[0185] (2) Rats' orbital blood sampling and serum anti-cyclic citrullinated peptide antibody (CCP-Ab) detection

[0186] The increase of ACPA in serum is a significant indicator of RA, and is an effective marker for judging whether the modeling is successful.

[0187] 1) Orbital blood sampling: On the 10th day and the 20th day after modeling, the Control group and the CIA modeling group were subjected to orbital blood sampling operation. According to the proportion standard, anesthetic was injected, the rats were placed on a flat operation table, the skin of the neck of the rat was pulled back with the left hand, the eyeball of the rat was protruded by this action, a sterile capillary glass tube (0.5*100mm) was placed at the inner canthus, inserted into the inner canthus at 30-45 degrees, inserted into the back of the eyeball, avoiding the eyeball, when the resistance was felt, the capillary was rotated, and then the blood flowed out, and the appropriate amount of blood was collected in a test tube without anticoagulant. The blood was left to stand at room temperature for 30-60 min, and then centrifuged at 3000 rpm for 10 min after the blood was coagulated. The supernatant serum was taken and stored in a-80℃ refrigerator.

[0188] 2) Serological detection:

[0189] a. The rat (Rat) anti-cyclic citrullinated peptide antibody Elisa kit was equilibrated at room temperature for 20 min, and the serum to be tested was thawed.

[0190] b. Preparation of washing solution: first prepare 20x washing buffer and distilled water, then slowly add 20x washing buffer into distilled water and mix well to obtain 1x washing buffer with appropriate concentration;

[0191] c. First, take out the plate, and add 50 μL of different concentrations of standards into each standard well. For sample wells, add 10 μL of serum to be tested, and then add 40 μL of sample diluent. Add 100 μL of HRP-labeled detection antibody to each reaction well to ensure accuracy and consistency of the amount added. After adding, quickly take the sealing film and tightly seal the reaction plate. Transfer the reaction plate to a constant temperature environment, such as a constant temperature incubator, at 37°C. Incubate the reaction plate in the constant temperature environment for 1 h.

[0192] d. After incubation, discard the liquid in the reaction plate. Then, wash the reaction plate with 1x washing buffer, and let the washing buffer act on the reaction plate for 1 min to thoroughly clean the residual impurities and unbound substances in the reaction plate. Then, gently shake the reaction plate to remove as much washing liquid as possible, and place the reaction plate upside down on absorbent paper to completely absorb the residual washing liquid. To ensure cleaning effect, repeat the above washing, shaking and absorbing operations five times to ensure that the reaction plate is thoroughly cleaned.

[0193] e. Add 50 μL of substrate A and B to each well, and incubate at 37°C for 15 min in the dark.

[0194] f. Add 50 μL of stop solution to each reaction well, and place at room temperature for 15 min, then place in a microplate reader. Set the wavelength of the microplate reader to 450 nm, and accurately measure the OD value of each reaction well.

[0195] g. The results are analyzed as follows: accurately mark the coordinate points formed by each concentration standard and its corresponding OD value on the coordinate graph, and draw the linear regression curve of the standard. After completing the curve drawing, calculate the concentration value of the related substance in the serum according to the obtained equation.

[0196] 4. Statistics and analysis

[0197] Quantitative data is expressed as mean ± SD, all data are subjected to t test, significance is calculated at 95% confidence interval, p<0.05 is considered statistically significant, *p<0.05, **p<0.01, ***p<0.001.

[0198] III. Experimental results

[0199] (1) Body weight and joint score of rats

[0200] After observation, it was found that after the first immunization, compared with the Control group, the rats in the CIA group had redness, swelling and infection at the injection site at the base of the tail, obvious redness and swelling at the paws, fever, and decreased mental activity. After the second immunization, compared with the Control group, the rats in the CIA group had acute swelling at the paws and joints, ulcers at the injection site, persistent lethargy, reduced appetite, decreased hair gloss, and slightly decreased body weight. For details, see Figure 4 -A. The joint index of the control group was 0 from 0 to 21 days after modeling. Compared with the control group, the arthritis index score of the CIA group increased after the first immunization, continued to increase after the second immunization, and the increase rate became larger, and then the increase rate gradually slowed down. For details, see Figure 4 -B.

[0201] (2) Detection of anti-cycloguanine peptide antibodies in rat serum

[0202] ELISA detection of rheumatoid autoantibody ACPA in the serum of each group found that the ACPA content in the serum of the Control group was always 0. Compared with the Control group, the ACPA content in the serum of the CIA group was significantly higher than that of the Control group. Figure 5 .

[0203] 4. Summary and Discussion

[0204] In medical research, animal disease models have become a key tool for in-depth investigation of disease mechanisms, progression, and subsequent treatment strategies. In the development of RA disease models, rats and mice are the most commonly used experimental animal species. Common modeling drugs include incomplete Freund's adjuvant, complete Freund's adjuvant, chicken collagen, and bovine collagen. These animal and drug selections offer significant advantages: First, their relative diversity facilitates research under relatively uniform conditions, minimizing interference caused by individual or drug-specific differences. Second, animals like rats and mice are easy to maintain and relatively inexpensive, while the aforementioned drugs are readily available and affordable. These economical and practical experimental materials provide a solid foundation for researchers and are a crucial prerequisite for the smooth advancement of RA research.

[0205] The use of SD rats to prepare the RA animal model in this embodiment has many advantages. From a practical perspective, SD rats are relatively convenient in experimental operations and can better meet research needs. At the same time, the model has good reproducibility, which means that the model establishment process can be reproduced relatively stably under the same experimental conditions. Its disease course lasts for a suitable period of time, providing a suitable time window for research. More importantly, the RA animal model constructed by SD rats has a high similarity with human rheumatoid arthritis in terms of pathological characteristics and pathogenesis, which is of great reference value for in-depth exploration of the relevant mechanisms of RA and evaluation of the effectiveness of treatment methods. During the modeling process, CⅡ type and CFA are mixed in a specific ratio and then injected into the rat body. By inducing the body to produce an immune response, the rat is prompted to exhibit the relevant pathological characteristics of rheumatoid arthritis, thereby achieving the purpose of constructing a rat model of the disease. This modeling success rate is high, and a model that meets research needs can be stably constructed, reducing the risk of experimental failure. At the same time, the cost required for this method is relatively low, which reduces research costs to a certain extent. Based on these advantages, this modeling method provides a solid and important experimental foundation for various clinical research projects, helping researchers to deeply explore the mechanisms of related diseases and evaluate the effectiveness of treatment plans. Clinical manifestations and serological tests were selected to verify the disease animal model. The results showed that compared with the control group, the CIA group had a slight decrease in body weight, clinical manifestations such as mental depression and limited activity, joint verification index ≥ 4 (n = 27), and the CCP content in the serum was significantly increased (p < 0.001). In summary, the RA disease animal model - CIA rat model - was successfully induced using bovine CⅡ and CFA drugs and SD rats.

[0206] Example 3. Evaluation of the therapeutic effect of hUC-MSCs on CIA rats

[0207] This study used a CIA rat model as the research subject, combining tail vein injection of hUC-MSCs with a positive drug. The study focused on observing the improvement of CIA symptoms in the rats and the effects on the expression of inflammatory factors in the rats. The goal was to further explore the actual effectiveness and anti-inflammatory mechanisms of hUC-MSCs in RA treatment, thereby providing experimental evidence for its subsequent widespread application in clinical practice.

[0208] 1. Experimental Materials

[0209] (1) Experimental animals

[0210] In Example 2, there were 7 blank control groups and 28 CIA rats with successful modeling.

[0211] (2) Preparation of main reagents

[0212] 1×PBS (0.01 M phosphate buffer): The preparation method is the same as Example 1.

[0213] 10% EDTA decalcification solution: Weigh 10g of EDTA and slowly add it to an appropriate amount of double-distilled water. Then, stir the solution thoroughly to ensure that the EDTA is completely dissolved in the double-distilled water. Adjust the pH of the solution to 7.2. Transfer the solution to a 100mL volumetric flask and dilute it to 100mL with double-distilled water. Finally, place the prepared solution in a dark environment and store it at room temperature for subsequent experiments.

[0214] The configuration system of 1×SDS-PAGE electrophoresis buffer is: Tris 3.02g, SDS 1g, glycine 18.8g, and ddH2O to 1L.

[0215] Protein gel transfer buffer: Tris 5.8g, SDS 0.37g, glycine 2.9g, double-distilled water to 800mL, methanol to 1L. After weighing these reagents, mix well before transferring or storing.

[0216] 10% ammonium persulfate (AP): Add 1 g of ammonium persulfate to 10 mL of ddH2O and mix well before use.

[0217] 5% SDS-PAGE stacking gel (2 mL): Double-distilled water 1.4 mL, 10% SDS 0.02 mL, 1 M Tris-HCl (pH 6.8) 0.25 mL, 10% ammonium persulfate 0.02 mL, 30% acrylamide 0.33 mL, TEMED 0.002 mL. Mix these reagents thoroughly and quickly add them to the gel sheet. Wait until the reagents in the gel sheet are completely solidified before use.

[0218] 10% SDS-PAGE separation gel (5 mL): double-distilled water 1.9 mL, 10% SDS 0.05 mL, 1.5 M Tris-HCl (pH=8.8) 1.3 mL, 10% ammonium persulfate 0.05 mL, 30% Acrylamide 1.7 mL, TEMED 0.002 mL.

[0219] 5x TBST buffer: 1M Tris-HCl (pH=8.0) 100ml, NaCl 44g, Tween20500ul. After all the reagents are accurately weighed, add double distilled water to make the total volume of the solution to 1L. Then, mix well with double distilled water, and store the solution at room temperature for later use.

[0220] PVDF membrane blocking solution: according to the use, weigh an appropriate amount of BSA reagent or skimmed milk powder, add TBST to a final concentration of 5%, mix well and use. Preferred: skimmed milk powder 2g, 1x TBST 40ml.

[0221] 4% paraformaldehyde: weigh an appropriate amount of paraformaldehyde, dissolve and dilute to a final concentration of 4% with PBS, mix well.

[0222] II. Experimental method

[0223] 1. Edu labeling of hUC-MSCs

[0224] (1) Preparation of EdU medium: take an appropriate amount of EdU stock solution, dilute it in cell culture medium according to the volume ratio of 1:1000 to prepare EdU medium with a concentration of 50umol / L. Then, add the same amount of cell culture medium to the medium to further ensure that the final concentration of EdU medium is stable at 50umol / L.

[0225] (2) EdU labeling of hUC-MSCs: on the second day after the third passage of hUC-MSCs, replace the cell growth medium in time. Then, accurately add the EdU solution prepared in step (1) with a concentration of 25umol / L to the culture dish, and continue to incubate the cells in this environment for 24h. After 24h, carefully discard the culture medium in the culture dish, wash the cells with PBS buffer treated by high pressure sterilization, each time for 5min, a total of 2 times, to completely remove the DNA that has not been successfully labeled with EdU, and ensure the specificity and accuracy of the labeling.

[0226] (3) Take hUC-MSCs with appropriate density, carefully remove the culture medium, and gently wash the cells with PBS buffer. After washing, add an appropriate amount of trypsin solution to the culture dish to promote cell digestion and dispersion into single cell state. When the cell digestion is complete, quickly add an appropriate amount of culture medium to terminate the trypsin digestion, and prepare a single cell suspension with a concentration of 1x10 7 7 ​The cell suspension was injected into the rats through tail vein injection at a dose of 1.0 x 10

[0227] 2. Experimental grouping and treatment

[0228] Twenty-eight CIA rats were randomly divided into four groups: a PBS injection and saline gavage treatment group (CIA group), an hUC-MSCs injection treatment group (CIA+MSC group), an MTX gavage treatment group (MTX group), and an hUC-MSCs injection and MTX gavage treatment group (MSC+MTX group).

[0229] CIA+MSC group: After the rats were anesthetized, they were placed on the operating table, and the tail was disinfected before being injected with 1.0 x 10 7 cells / kg (rat weight) of hUC-MSCs suspension through tail vein injection.

[0230] CIA+MTX group: The MTX gavage operation procedure was performed, and the administration frequency was set to once a week, with the administration dose controlled at 5 mg, and the administration was performed regularly

[0231] MSC+MSC+MTX group: The CIA rats were injected with hUC-MSCs suspension and treated with MTX in combination using the same method and dose.

[0232] CIA group: The CIA rats were injected with PBS through tail vein injection and gavaged with the same dose of saline using the same method and dose.

[0233] Control group: The normal rats were gavaged with the same dose of saline using the same method and dose.

[0234] After the injection operation was completed, the needle was slowly pulled out of the injection site, and the injection site was then appropriately compressed to achieve hemostasis. After the hemostasis operation was completed, the rats were returned to the original cages and continued to be raised according to the normal raising procedure.

[0235] 3. Observation of the body weight and clinical manifestations of the rats

[0236] Starting from the first day after treatment, the rats were monitored for a series of indicators: 1) Weight changes were recorded. Weight fluctuations can reflect the rat's overall physical condition and the potential impact of treatment on its metabolism. 2) The luster of the rats' hair was carefully observed. The quality of the hair is closely related to the animal's health level, and a dull luster may indicate poor health. 3) The redness and swelling of the rats' joints and paws were observed. Joint redness and swelling are key external indicators of RA severity, and increasing or decreasing redness and swelling are crucial for understanding the progression of the disease. 4) The pathological changes in the rats' joints were further investigated. Details such as joint deformity and bone destruction provide key information for accurate disease assessment. Simultaneously, the condition of the rats' limbs was rigorously and meticulously scored according to the pre-established Arthritis Index scoring criteria shown in Table 3.

[0237] 4. HE staining of knee joint

[0238] (1) Knee joint decalcification: After 4 weeks of treatment, the rats in each group were sacrificed.

[0239] Then, the left ankle joint of the rat was removed and the muscles and other soft tissues attached to it were carefully removed. The treated ankle joint was placed in a pre-prepared 4% neutral paraformaldehyde solution for fixation. Next, the joint was placed in a 1:10 volume of EDTA decalcification solution for decalcification at room temperature.

[0240] Decalcify for 20-30 days, and replace the EDTA decalcification solution every 5-7 days depending on the situation.

[0241] (2) HE staining:

[0242] a. When the knee joint can be easily punctured by a needle, perform routine dehydration and wax impregnation according to the following procedures:

[0243] 75% ethanol for 30 min; 85% ethanol for 30 min; 95% ethanol for 30 min; 95% ethanol for 30 min; anhydrous ethanol for 30 min; anhydrous ethanol for 30 min; xylene I for 30 min; xylene II for 30 min; paraffin overnight.

[0244] b. Dewax and hydrate according to the following procedures:

[0245] Xylene I, 5 min; Xylene II, 5 min; 1 / 2 xylene, 5 min; Anhydrous ethanol I, 5 min; Anhydrous ethanol II, 5 min; 95% ethanol, 5 min; 85% ethanol, 5 min; 75% ethanol, 5 min; ddH2O, 5 min.

[0246] c. Stain the sample with hematoxylin for 7-10 minutes. After staining, rinse thoroughly with running water to remove any residual hematoxylin solution. Next, perform a color separation on the sample using a 1% hydrochloric acid-ethanol solution for 10 seconds. Rinse the sample with running water for 20 seconds, followed by a bluing treatment with ammonia solution for another 20 seconds. After the bluing is complete, rinse the sample again with running water and observe the blue color of the cell nuclei under a microscope.

[0247] d. Place in eosin solution for 3 minutes and rinse with water for 2 seconds.

[0248] e. The dehydration and sealing steps are as follows: first soak the sample in 75% ethanol solution for 2 minutes, then move it to 80% ethanol solution and continue soaking for 2 minutes, then soak it in 95% ethanol solution for 2 minutes, then place the sample in anhydrous ethanol, soak it for the first time for 1 minute, and then soak it in anhydrous ethanol for the second time, also for 1 minute. After completing the ethanol dehydration step, soak the sample in xylene I and xylene II successively, each soaking time is 1 minute. Use neutral gum to seal the sample. After sealing, place the sample under a microscope for detailed observation. At the same time, take photos of the observed results for subsequent analysis and processing of the captured images.

[0249] f. Record histopathological changes and score the degree of joint inflammation and joint destruction according to Table 3.

[0250] Table 3. Scoring criteria for joint tissue inflammation and changes

[0251] Score Pathology scoring principles 0 No histopathological changes 1 Mild focal infiltration of inflammatory cells 2 Neutral infiltration of inflammatory cells 3 Neutral infiltration of inflammatory cells 4 Neutral infiltration of inflammatory cells

[0252] 5. Masson staining of joints

[0253] a. Dewax and hydrate the tissue as above.

[0254] b. Nuclear Hematoxylin Staining: Use Weigert's iron hematoxylin reagent from the Masson staining kit to stain the cell nuclei for 5 minutes. After staining, rinse the stained sample with tap water to remove excess staining reagent. Then, place the sample in a 1% hydrochloric acid-alcohol solution for a few seconds to differentiate. After differentiation, rinse the sample again with tap water to remove the hydrochloric acid-alcohol solution. Finally, rinse the sample in running water for several minutes. The running water will cause the cell nuclei to return to a blue color, achieving the desired staining state.

[0255] c. Ponceau Staining Procedure: Use the Ponceau Acid Fuchsin solution from the Masson Staining Kit to stain the sample for 5-10 minutes. Immediately rinse with ddH2O.

[0256] d. Phosphomolybdic acid treatment step: The sample is completely immersed in the solution. The treatment time is controlled between 3-5 minutes to ensure that the sample can fully react with the phosphomolybdic acid aqueous solution.

[0257] e. Aniline Blue Staining: Without washing, counterstain the sample directly using the aniline blue solution in the Masson staining kit. Completely immerse the sample in the aniline blue solution to ensure full contact between the sample and the stain. Strictly control the counterstaining time to 5 minutes to ensure the staining effect meets experimental requirements and provides accurate sample status for subsequent observation and analysis.

[0258] f. Differentiation stage: Use 1% glacial acetic acid solution to differentiate the sample. Place the sample in the glacial acetic acid solution, ensuring that it is completely immersed. The treatment time is precisely controlled to 1 minute.

[0259] g. Seal the slides with neutral gum as above, observe under a microscope, take photos and save the images for analysis.

[0260] 6. Safranin O-Fast Green Staining of Knee Joint

[0261] a. Deparaffinize according to the normal procedure until the sample is hydrated.

[0262] b. Place the sample to be processed into the freshly prepared Weigert stain solution, ensuring that the sample is completely immersed in the stain solution, and stain for 3-5 minutes.

[0263] c. Then transfer it to acidic ethanol differentiation solution and control the differentiation time to 15 seconds.

[0264] d. After the sample is differentiated, place it in distilled water. Ensure the sample is completely submerged in the distilled water and rinse for 1 minute. This rinse will effectively remove any excess reagent from the sample surface and interior.

[0265] e. Immerse the sample in Fast Green staining solution for 5 minutes, then rinse with distilled water for 1 minute.

[0266] f. Then place the sample in Safranin 0 dye solution and soak for 1-2 minutes. Rinse with distilled water for 1 minute.

[0267] g. Wash the sections with acetic acid for 1-2 minutes to remove any remaining fast green. Rinse with distilled water for 1 minute.

[0268] h. Dehydrate the sample using 95% ethanol and then anhydrous ethanol, then make it transparent using xylene. Finally, seal the slide with neutral gum. Observe the experimental results under a microscope and photograph the resulting image for subsequent analysis.

[0269] 7. Elisa method for detecting inflammatory factors

[0270] The blood of each group of rats was collected in a test tube to extract serum, which was stored at -80°C. The levels of IL-10, TNF-α, CCP, etc. in the serum of each group of rats were detected in the same manner as in Example 2, "rat orbital blood collection and serum anti-cycloguanine peptide antibody detection".

[0271] 8. Western blot

[0272] (1) Preparation of protein samples

[0273] a. Protein extraction: Place a small amount of joint tissue in tin foil, quickly freeze it in liquid nitrogen, and then grind it thoroughly in an autoclaved mortar. Place the ground tissue in a centrifuge tube.

[0274] b. Accurately add 200μl of lysis buffer to each sample tube. The lysis buffer contains 2μl of PMSF and 2μl of phosphatase inhibitors to ensure the lysis effect and prevent abnormal changes in protein phosphorylation. After the sample is added, quickly place the sample tube on ice and allow the lysis buffer to fully react with the sample in a low temperature environment for 40 minutes. During this period, the sample tube can be gently shaken to ensure that the sample can be fully lysed.

[0275] c. Place the tube in a centrifuge at 4°C, 12,000 rpm, and 5 minutes. After centrifugation, remove the supernatant and aliquot into 0.5 ml centrifuge tubes.

[0276] (2) Determination of protein concentration

[0277] a. Melt the standard sample protein slowly on ice and prepare the BCA reagent.

[0278] b. Prepare a 96-well plate and add the protein standard solution according to the gradient concentration. Add PBS to a final volume of 20 μL. Add 1 μL of the protein sample to the well with the test protein, followed by 19 μL of PBS. Finally, add 200 μL of the prepared BCA reagent to each well. Repeat for each sample in triplicate.

[0279] c. Incubate in 65 °C incubator for 6-10 min.

[0280] d. Add protein samples into the corresponding wells of the microplate reader, set the wavelength of the microplate reader to OD 560, record the data, and calculate the protein sample concentration.

[0281] (3) Preparation of experimental samples

[0282] According to the standard curve, calculate the concentration of the protein sample to be tested, configure the protein sample according to the requirements, then add loading buffer to it according to the specific requirements of the experiment, mix well, heat at 96 °C for 10 min, and save the sample or proceed to the next step according to the experimental requirements.

[0283] (4) Polyacrylamide gel electrophoresis (SDS-PAGE): Prepare protein concentration gel and separation gel, add gel plate, and perform electrophoresis after gelation. Electrophoresis parameters: constant current electrophoresis at 25 mA.

[0284] (5) Membrane transfer

[0285] a. Soak the cut PVDF membrane in methanol solution for activation, so that it can better perform in subsequent experiments. The soaking time is about 1 min, which prepares for subsequent experimental operations such as protein transfer.

[0286] b. Add an appropriate amount of buffer to the membrane transfer box, place the filter paper pad, PVDF membrane, protein gel, and filter paper pad in the "sandwich" model in order. Ensure that each layer is tightly attached and free of air bubbles to ensure smooth membrane transfer. After placement, place the membrane transfer instrument into the membrane transfer instrument for membrane transfer. Membrane transfer parameters: 25V, 1A, 30min.

[0287] (6) Blocking and exposure

[0288] a. Prepare TBST blocking solution containing 5% skimmed milk powder.

[0289] b. After the membrane transfer operation is completed, immerse the PVDF membrane in the blocking solution and perform blocking treatment for 2h.

[0290] c. According to the requirements, cut the target protein band and immerse it in the primary antibody for 4 °C overnight.

[0291] d. Recover the primary antibody, wash with TBST for 3x5min, and after washing, add the corresponding secondary antibody solution and incubate at room temperature for 2h.

[0292] f. Recover the secondary antibody, wash with TBST for 3x10min, and after washing, perform exposure.

[0293] III. Experimental results

[0294] (1) Body weight and arthritis score of rats

[0295] The results are shown in Table 4, Figure 6 and Figure 7 After treatment, the body weight of the Control group gradually increased over time, the joints were not swollen, and the arthritis score was 0. Compared with the Control group, the appetite of the CIA group rats decreased, the body weight increased slowly, the whole joint was red and swollen, reached the state of being unable to walk, and the arthritis score was the highest. In the CIA+MSC group, the body weight increased, the degree of joint swelling gradually decreased during treatment, and the arthritis score decreased. In the CIA+MTX group, there was no significant difference in body weight, the degree of joint swelling decreased slightly, and the arthritis score decreased slightly. In the CIA+MSC+MTX group, the body weight was higher than that of the CIA group, the degree of joint swelling decreased significantly, and the arthritis score decreased significantly.

[0296] Table 4. Body weight change table of rats in each group Compared with the CIA group, *P≤0.05, **P≤0.01

[0297]

[0298] (2) EdU fluorescence staining

[0299] After completing the EdU staining operation, the cell proliferation detection work was immediately carried out with the help of a fluorescence microscope to observe the proliferation dynamics of stem cells. In order to more clearly present the distribution details of the relevant cells in the joint tissue, the joint tissue was dyed with a cell nucleus specific dye Hoechst. After this dyeing, the blue fluorescence was accurately positioned in the cell nucleus region, which could clearly outline the outline of the cell nucleus. Then, the fluorescence image presented by EdU staining and the image obtained by Hoechst staining were overlapped and integrated, so that the colonization situation of the labeled hUC-MSCs in the joint tissue could be directly observed. Specifically, according to the observed fluorescence depth and the corresponding cell number, the actual number of human umbilical cord mesenchymal stem cells in the tissue could be effectively reflected. The experimental results are shown in Figure 8 and Figure 9 After intravenous injection of EdU-labeled hUC-MSCs, colonization of the cell population in the joint at different degrees was observed at the time nodes of 3 days and 7 days.

[0300] (3) HE staining of knee joint

[0301] Pathological analysis of joint tissue: The joint structure of the Control group was intact and clear, with no joint damage, uniform cell distribution, and no inflammatory cell infiltration. Compared with the Control group, the joint structure of the CIA group was significantly damaged, with chaotic and uneven cell distribution and a large number of lymphocyte infiltration. Compared with the CIA group, the CIA+MSC group had a more uniform cell distribution, a slight improvement in lymphocyte infiltration model group, and a decreased joint tissue score. In the CIA+MTX group, the cell distribution was more uniform, lymphocyte infiltration was slightly improved, and the joint tissue score decreased, but the degree of infiltration was slightly greater than that of CIA+MSC. In the CIA+MSC+MTX group, the structure was intact, the cells were uniformly distributed, the degree of lymphocyte infiltration was significantly improved, and the joint tissue score decreased significantly. For details, see Figure 10 -A.

[0302] Joint tissue injury score: The joint tissue score of the Control group was 0, and the joint injury scores of CIA+MSC and CIA+MTX were lower than those of the CIA group. Moreover, the joint tissue score of CIA+MSC+MTX was significantly lower than that of the CIA group. Figure 10 -B. The results showed that compared with the CIA group, the CIA+MSC group and CIA+MTX group could alleviate the inflammatory cell infiltration in the rat joints, among which the CIA+MSC+MSC group had the most obvious effect.

[0303] (4) Masson staining of joints

[0304] Masson staining results Figure 11 The results showed that: Masson staining in the control group was normal, with mature bone tissue maintaining red staining; compared with the control group, the CIA group showed significant joint bone tissue destruction, with newly formed bone stained blue by toluidine blue and severe loss of mature bone tissue, which was stained red by Ponceau red; compared with the CIA group, the red staining area in the CIA+MSC, CIA+MTX, and CIA+MSC+MTX groups increased significantly, demonstrating that each treatment group had a certain ability to repair damaged bone tissue in rats; the CIA+MSC+MTX group had a slightly higher repair capacity than the other treatment groups, alleviating joint damage in CIA and demonstrating significant joint repair capabilities.

[0305] (5) Safranin O-Fast Green staining of joints

[0306] Safranin O-Fast Green staining is a staining method that can visually present the characteristics of tissues such as articular cartilage. During the staining process, cartilage with basophilic characteristics will combine with the basic dye Safranin O, thereby appearing red; while bone tissue with eosinophilic characteristics will react with the acidic dye Fast Green to appear blue. The results of Safranin O-Fast Green staining clearly show that: the articular cartilage structure of the Conrtol group is intact; compared with the Conrtol group, the articular cartilage structure of the CIA group was significantly damaged; compared with the CIA group, the cartilage tissue of the CIA+MSC group, CIA+MTX group and CIA+MSC+MTX group was significantly repaired, indicating that the treatment of each group can effectively promote cartilage repair; among them, the CIA+MSC+MTX group represents the largest red part of the cartilage and has the strongest ability to repair articular cartilage, followed by the CIA+MSC group and CIA+MTX group, see for details. Figure 12 .

[0307] (6) Elisa method for detecting inflammatory factors

[0308] ELISA assay showed that the levels of ACPA, proinflammatory factors IL-6, and TNF-α in the serum of rats in the CIA group were significantly higher than those in the control group (P < 0.0001). Compared with the CIA group, the levels of CCP, proinflammatory factors IL-6, and TNF-α in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group were significantly decreased, and the decrease in proinflammatory factors in the CIA+MSC+MTX group was more significant (p ≤ 0.0001). Figure 13 -A. Figure 13 -B and Figure 13 -C.

[0309] The level of anti-inflammatory factor TGF-β in the serum of rats in the CIA group was higher than that in the Control group (P < 0.005), while there was no significant difference in IL-10 level. The levels of IL-10 and TGF-β in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group were significantly higher than those in the Control and CIA groups (P < 0.01), and the levels in the CIA+MSC+MTX group were the highest (p ≤ 0.001 or p ≤ 0.0001). Figure 13 -D and Figure 13 -E.

[0310] (7) Western blot

[0311] To further verify the therapeutic effects of hUC-MSCs, MTX, and hUC-MSCs and MTX on CIA rats, we performed Western blotting experiments. Figure 14As shown in the results, compared with the CIA group, the levels of related pro-inflammatory factors IL-6 and TNF-α in the three groups were significantly decreased, and the levels in the CIA+MSC+MTX group were the lowest; the pro-inflammatory factors IL-10 and TGF-β were highly expressed, and the levels in the CIA+MSC+MTX group were the highest.

[0312] 4. Summary and Discussion

[0313] A series of staining experiments were conducted on joint tissue samples of rats in different groups. The results showed that hUC-MSCs, MTX, and the combination of the two all showed significant improvement in the lymphocyte infiltration of CIA rats, and had the ability to repair bone tissue and cartilage tissue. Among these treatment methods, the effect of hUC-MSCs combined with MTX was particularly outstanding. At the same time, the serum cytokines of rats in each group were further detected, and with the help of protein immunoblotting experiments, quantitative analysis of related factors in the joint proteins of each group was carried out. The results showed that in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group, the expression levels of related pro-inflammatory factors decreased, and the expression levels of anti-inflammatory factors were increased.

[0314] Based on the above experimental results, it can be seen that hUC-MSCs and MTX have significant therapeutic effects on CIA rats, among which the therapeutic effect when hUC-MSCs and MTX are used in combination is the most significant.

[0315] Example 4. Effect of hUC-MSCs on synovial macrophage polarization

[0316] In the complex pathological process of RA, synovial inflammation is a key component, not only in the production of autoantibodies and the fluctuating levels of related inflammatory factors. Numerous current research findings point to a key conclusion: a strong correlation between synovial inflammation and macrophage polarization. It is now clear that activated macrophages primarily exhibit two polarization types: M1 and M2. Typical markers of M1 macrophages include CD86 and inos. M1 macrophages secrete large amounts of pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), driving the inflammatory response. In contrast, M2 macrophages, characterized by markers such as CD163, tend to secrete large amounts of factors that suppress inflammation. Given the crucial role of macrophage polarization and its underlying mechanisms in the progression of RA, research in this area has been a hot topic in the academic community. This experiment comprehensively used multiple experimental methods such as qRT-PCR experiments, Western blot, and IHC to explore the polarization state of synovial macrophages, striving to deeply explore the specific effects of macrophage polarization on synovial inflammation.

[0317] 1. Experimental Animals

[0318] In Example 2, there were 7 blank control groups and 28 CIA rats with successful modeling.

[0319] 2. Experimental Methods

[0320] 1. Acquisition of synovial tissue

[0321] The rat was immersed in 75% alcohol for 10 minutes and then fixed in the supine position on a laboratory table. A longitudinal skin incision was made along the midline of the knee joint, exposing an area approximately 3 cm x 3 cm centered on the knee joint. The incision began approximately 0.3-0.4 cm above the superior edge of the patella and was continued in the designated direction until the femoral surface was reached. Subsequently, the patella was dissected downward along both sides until the tibia was reached. The knee joint cavity was now open, revealing a layer of smooth, shiny, pale yellow synovial tissue extending upward from the inferior pole of the patella. During dissection, the synovial tissue was carefully dissected intact. Finally, the free end of the synovial tissue was carefully grasped with ophthalmic forceps and completely excised with a razor blade. The tissue was fixed with paraformaldehyde, embedded, and sectioned for pathological examination or washed with sterile PBS and stored at -80°C for protein and RNA extraction.

[0322] 2. RNA Extraction and qRT-PCR

[0323] (1) RNA extraction

[0324] 1) Take out the preserved synovial tissue sample.

[0325] 2) Add 200 μL of chloroform to the sample and shake vigorously for 15 seconds. After shaking, place the sample on ice for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes.

[0326] 3) After centrifugation, transfer the supernatant to a new centrifuge tube, add an equal amount of isopropanol, mix thoroughly, and let it stand at room temperature for 2 minutes.

[0327] 4) Centrifuge at 12000 rpm for 5 min, discard the supernatant, add 1 mL of pre-cooled anhydrous ethanol to the precipitate, gently blow up the precipitate, and let it stand at room temperature for 2 min.

[0328] 5) Centrifuge the sample at 7500 rpm for 5 minutes. Discard the supernatant. Allow the sample to air dry at room temperature for 5-10 minutes. After air-drying, add 30-50 μL of RNAase-free water. Assay the RNA concentration and refrigerate until ready for the next step.

[0329] (2) Synthesis of cDNA template chain

[0330] Use the YEASEN Biotech Reverse Transcription Kit to synthesize the cDNA template strand. For details on the transcription system and protocol, refer to the kit instructions. After reverse transcription, store the product as needed or proceed to the next step.

[0331] (3) qRT-PCR

[0332] 1) Based on the gene sequences of CD86, CD163, iNOS, IL-6, IL-10, and GADPH, quantitative primer sequences were designed. qRT-PCR experiments were performed using previously prepared cDNA as templates. The quantitative primer sequences used in this study are shown in Table 5.

[0333] Table 5. Sequence list of quantitative primers

[0334]

[0335]

[0336] 2) The qRT-PCR system included: Mix, 10 μL; double-distilled water, 7 μL; cDNA, 2 μL; upstream primer, 0.5 μL; downstream primer, 0.5 μL; total volume: 20 μL. The qRT-PCR kit was from YEASEN Biotechnology.

[0337] 3) After adding the sample, centrifuge and mix thoroughly. Set the quantitative PCR program as follows: pre-denaturation at 95°C for 5 minutes, 1 cycle; denaturation at 95°C for 15 seconds, 45 cycles; annealing and extension at 60°C for 30 seconds.

[0338] 4) After the PCR process is completed, the obtained data are analyzed using the relative quantification method.

[0339] (4) Western blot: same as in Example 3.

[0340] (5) Immunohistochemical staining

[0341] Tissue fixation is the same as above.

[0342] 1) Sectioning operation: Fix the embedded synovial tissue on the base of the paraffin slicer and adjust the section thickness to 2 μm.

[0343] 2) Slice operation: Add double distilled water into the slice machine in advance, and set the temperature of the slice machine to 42°C. Put the cut slices into a water bath. When the slices are fully expanded in the water bath, use a glass evenly coated with egg white to pick up the slices. After the water on the glass is dried, store it in a 4°C environment

[0344] 3) Toasting operation: Place the prepared slices neatly on the shelf, then put the shelf into the oven with a temperature setting of 65°C for 3-4h to make the paraffin around the synovial membrane tissue drop.

[0345] 4) De-waxing: De-waxing is carried out in the following order: dimethylbenzene (I), 10 min; dimethylbenzene (II), 10 min; 1 / 2 dimethylbenzene, 10 min; anhydrous ethanol (I), 5 min; anhydrous ethanol (II), 5 min; 95% alcohol (I), 5 min; 95% alcohol (II), 5 min; 85% alcohol, 5 min; 75% alcohol, 5 min.

[0346] 5) Nuclear protein Triton X-100 (0.5%) punch 1h. Membrane proteins can skip this step.

[0347] 6) Antigen repair (microwave repair)

[0348] The antigen repair process includes: microwave heating of the antigen repair solution, twice, each time for 5 min. After the antigen repair solution is heated to boiling, the slices are placed in it, and then microwave heating is continued for 5 min. After heating, the slices are removed from the repair solution and placed in a room temperature environment to cool naturally, with a cooling time of 7-8 min. Then, the slices and repair solution are again microwave heated for 5 min. Finally, cool to room temperature.

[0349] 7) Incubate the sample with 3% hydrogen peroxide solution for 10 min. Pay special attention to avoid light during the entire incubation process.

[0350] 8) PBST (add appropriate amount of Triton X-100 (0.1%) and tween 20 in PBS) wash 3x5min.

[0351] 9) 5% goat serum (diluted with 5% BSA) blocking 1h.

[0352] 10) Primary antibody 4°C incubation overnight.

[0353] 11) PBST wash 3x5min

[0354] 12) Carefully add the reaction enhancer (reagent II) to the sample, ensuring that the liquid covers the sample evenly. Then, incubate the sample at room temperature for 20 minutes.

[0355] 13) Wash with PBST 3 x 5 minutes.

[0356] 14) Accurately pipette the enhancer enzyme goat anti-rabbit / mouse IgG polymer onto the sample, and slowly drop it onto the sample to ensure that the liquid spreads evenly. Then, incubate the sample at room temperature for 20 minutes.

[0357] 15) Wash with PBST 3 x 5 minutes.

[0358] 16) Perform the DAB color development operation: Take 1 ml of the base solution, and accurately add 1 drop of reagent (DAB concentrate) to it. Mix or shake the two solutions thoroughly to ensure that the DAB concentrate is completely dispersed in the base solution, ready for subsequent color development of the sample.

[0359] 17) When the DAB color development step is complete, and the sample has developed the desired color, quickly place the sample under running tap water to terminate the color development reaction, ensuring that the color development does not continue to change.

[0360] 18) Hematoxylin counterstaining: 6-8 minutes (adjust according to the number of times the hematoxylin is used).

[0361] 19) Perform differentiation and blueing: First, prepare a 1% hydrochloric acid solution using 70% ethanol, and place the sample in the hydrochloric acid solution for 7 seconds. After differentiation, rinse the sample with tap water. Then, prepare a blueing solution by adding 1-2 drops of ammonia water to a container of water, and place the rinsed sample in the blueing solution for about 1 minute to achieve blueing. After the operation is complete, observe the sample under a microscope to determine the staining level. If the staining is too light, re-stain the sample. If the staining is too dark, perform differentiation again.

[0362] 20) Dehydration: Dehydrate the sample according to the following steps.

[0363] 75% alcohol for 5 minutes; 85% alcohol for 5 minutes; 95% alcohol (II) for 5 minutes; 95% alcohol (I) for 5 minutes; anhydrous ethanol (II) for 5 minutes; anhydrous ethanol (I) for 5 minutes; 1 / 2 xylene for 5 minutes; xylene (II) for 5 minutes; xylene (I) for 5 minutes.

[0364] 21) Mount the sample with neutral balsam, and use the microscope's built-in photographing equipment to take pictures of the sample, and save the images for future viewing and analysis.

[0365] III. Experimental Results

[0366] (1) Analysis of macrophage marker expression in synovial tissue

[0367] RT-qPCR and Western blot were used to detect the mRNA expression levels of M1 macrophage surface markers CD86 and iNOS and related pro-inflammatory factor IL-6; the mRNA expression levels of M2 macrophage surface marker CD163 and related anti-inflammatory factor IL-10, and the ratio of CD163 / CD86 in synovial tissue. Figure 15 、 Figure 16 As shown in the data, compared with the CIA group, the expression levels of CD86 and IL-6 decreased, the expression levels of CD163 and IL-10 increased, and the CD163 / CD86 ratio increased in the CIA+MSC+MTX group, CIA+MTX group, and CIA+MSC+MTX group. The results of the CIA+MSC+MTX group were more significant, indicating that all treatment groups could transform the pro-inflammatory M1 type of macrophages into the anti-inflammatory M2 type, the content of pro-inflammatory factors decreased, and the content of anti-inflammatory factors increased, and the combination of hUC-MSCs and MTX had the best effect.

[0368] (2) Synovial histochemical staining

[0369] IHC staining was used to verify the macrophage marker F4 / 80. The staining showed that the F4 / 80 positive rate in the synovial tissue of the CIA group was significantly increased, with more activated macrophages and synovial inflammation. After treatment, the F4 / 80 positive marker decreased, with the CIA+MSC+MTX group having the lowest positive rate, and the difference was statistically significant (P<0.001). Figure 17 Compared with the Control group, the CD86 positive rate in the CIA group was significantly increased, and the CD163 positive rate was significantly decreased. Compared with the CIA group, the CD86 positive rate in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group was significantly decreased, and the CD163 positive rate was significantly increased, with the CIA+MSC+MTX group being the most significant. Figure 18 The trend is the same as that of “Analysis of macrophage marker expression in synovial tissue”.

[0370] In summary, the treatment group inhibited synovial inflammation in CIA rats and promoted the transformation of synovial macrophages from the M1 type that promotes inflammation to the M2 type that inhibits inflammation.

[0371] 4. Summary and Discussion

[0372] Macrophages, as a kind of immune cells, have significant heterogeneity on their surface. When stimulated by various signals from different microenvironments, macrophages can polarize and form various cell phenotypes: M1 macrophages and M2 macrophages. M1 macrophages have strong pro-inflammatory effects and can secrete a large amount of pro-inflammatory cytokines such as TNF-α, IL-1β, etc., participating in immune defense and inflammatory response; M2 macrophages have anti-inflammatory and pro-tissue repair functions, can secrete anti-inflammatory cytokines such as IL-10, and some growth factors such as vascular endothelial growth factor (VEGF), which are beneficial to wound healing and tissue remodeling. In the process of this experimental research, in order to explore the polarization state of synovial macrophages, qRT-PCR experiments, Western blotting, immunohistochemistry, etc. were used. The results of the exploration show that hUC-MSCs and MTX both play a specific role in the exploration of macrophage polarization. Specifically, both can promote the transformation of the polarization direction of macrophages, prompting the M1 macrophages with pro-inflammatory properties to gradually transform into M2 macrophages with anti-inflammatory ability to inhibit inflammation. When hUC-MSCs and MTX are used in combination, the transformation promotion effect reaches the most significant degree, providing a solid basis for subsequent in-depth research and the development of clinical treatment strategies for related diseases.

[0373] Example 5. Exploration of the mechanism of rheumatoid arthritis

[0374] The research results of the foregoing examples show that hUC-MSCs have good effect in anti-RA inflammation, significantly reducing the levels of pro-inflammatory factors and significantly increasing the levels of anti-inflammatory factors in the serum and tissues of CIA rats. However, the molecular action of hUC-MSCs in RA treatment has not been clearly defined. The NF-κB signaling pathway is closely related to the pathogenesis of RA. The NF-κB signaling pathway not only activates inflammation in the body but also produces an apoptotic effect. The Bax / Bcl-2 signaling pathway has the greatest impact on apoptosis, so the interaction between inflammation and apoptosis accelerates the course of RA. This study will explore whether hUC-MSCs can inhibit the occurrence of inflammation through the NF-κB inflammation-related signaling pathway and regulate the Bax / Bcl-2 signaling pathway to achieve anti-apoptotic effects, thereby alleviating joint damage and having a therapeutic effect on RA. The expression of related signaling pathway-related targets will be studied to explore the related mechanism of hUC-MSCs as a potential diagnostic and therapeutic target for RA.

[0375] I. Experimental animals

[0376] In Example 2, 7 rats in the blank control group (Control group) and 28 CIA rats successfully modeled.

[0377] II. Experimental methods

[0378] (1) Harvesting of synovial tissue: same as Example 4.

[0379] (2) Western blot: same as Example 3.

[0380] (3) RNA extraction and qRT-PCR: same as Example 4.

[0381] The quantitative primer sequences were designed according to the gene sequences of p65, p-65, Bax, Bcl-2 and a-Tublin. The qRT-PCR experiments were performed using the previously prepared cDNA as the template. The quantitative primer sequences used in this paper are as follows in Table 6:

[0382] Table 6. Table of quantitative primer sequences

[0383]

[0384] (4) TUNEL detection of synovial tissue

[0385] 1) De-waxing and hydration treatment for paraffin tissue sections.

[0386] a. Permeation step: Proteinase K solution with a concentration of 2 mg / mL was diluted with PBS at a dilution ratio of 1:50 to obtain a final concentration of 40 μg / mL. Then, 100 μL of the diluted solution was added to the surface of each sample to ensure that the solution completely covered the sample area. Then, the sample was placed in a constant temperature environment at 37°C for continuous incubation for 30 min.

[0387] b. After the incubation was completed, the section sample was rinsed with 1x PBS solution for 3x 5 min. After rinsing, the excess liquid on the surface of the sample was gently absorbed with filter paper, and then the treated sample was placed in a wet box to maintain the wet state of the sample.

[0388] 2) TUNEL reaction

[0389] Preparation of TUNEL reaction solution (use immediately after preparation):

[0390] a. Add 50 μL of TUNEL reaction solution to each sample to ensure that the reaction solution uniformly covers the entire sample. Then incubate the sample at 37°C in the dark for 1 h.

[0391] b. After incubation, discard the TUNEL solution from the sample. Wash the sample twice with PBS, then three times with 0.1% Triton X-100 (prepared in PBS and containing 5 mg / mL BSA), each wash lasting 5 minutes. This procedure will more thoroughly remove free, unreacted markers.

[0392] c. Add an appropriate amount of 5 μg / mL DAPI stain to each sample and incubate for 5 minutes at room temperature in a dark environment. After staining, discard the DAPI stain and rinse the sample with PBS for 5 minutes each time.

[0393] d. Perform sectioning and sealing operation: add 20 μL of anti-fluorescence quenching sealing medium to each sample.

[0394] e. Use filter paper to remove excess liquid from the sample, then add 100 μL of PBS to the sample area to keep the sample moist, and then immediately observe the sample under a fluorescence microscope.

[0395] 3. Experimental Results

[0396] (1) Western blot and qRT-PCR analysis

[0397] The results were detected by qRT-PCR and Western blot. Figure 19 The results are shown in Table 7. The results showed that the expression of NF-κB and Bax / Bcl-2 apoptosis signaling pathways in each treatment group showed a significant increase in the expression level of p-p65 protein in the CIA group compared with the control group (p < 0.01). Compared with the CIA group, the expression of p-p65 protein in the CIA+MSC group and CIA+MSC+MTX group was significantly downregulated (P < 0.05), and there was no significant difference in the CIA+MTX group. Compared with the control group, the expression of p-p65 / p65 in the CIA group was significantly increased (p < 0.0001). Compared with the CIA group, the expression of p-p65 / p65 in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group was significantly downregulated (P < 0.05 or P < 0.001).

[0398] Compared with the Control group, the expression of Bax protein in the CIA group showed a very significant upward trend (P < 0.0001); compared with the CIA group, the expression of Bax protein in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group was significantly downregulated (P < 0.01 or P < 0.05); compared with the Control group, the expression of Bcl-2 protein in the CIA group showed a significant downregulation trend (P < 0.001); compared with the CIA group, the expression of Bcl-2 protein in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group was significantly upregulated (P < 0.05 or P < 0.001); compared with the Control group, the expression of Bax / Bcl-2 in the CIA group was significantly increased (P < 0.001); compared with the CIA group, the expression of Bax / Bcl-2 in the CIA+MSC group, CIA+MTX group, and CIA+MSC+MTX group was significantly downregulated.

[0399] Table 7. p65, p-p65, Bax and Bcl-2 mRNA expression and analysis results

[0400] p65 p-p65 p-p65 / p65 Bax Bcl-2 Bax / Bcl-2 Control 0.73±0.03 0.47±0.15*** 0.64±0.10*** 0.46±0.05*** 0.80±0.12*** 0.57±0.13*** CIA 0.71±0.05 1.26±0.17 1.77±0.03 0.85±0.07 0.33±0.07 2.57±0.02 CIA+MSC 0.67±0.03 0.81±0.10* 1.20±0.10** 0.56±0.08** 0.30±0.05* 1.86±0.06* CIA+MTX 0.74±0.07 0.91±0.12* 1.22±0.11** 0.62±0.09* 0.78±0.06* 0.79±0.08* CIA+MSC+MTX 0.73±0.06 0.67±0.16** 0.91±0.15** 0.56±0.12** 0.64±0.07** 0.87±0.05**

[0401] (2) TUNEL assay for cell apoptosis

[0402] The TUNEL method was used to detect the cell apoptosis in synovial tissue and the results were as follows. Figure 20 Normal cells appear blue, while apoptotic cells appear fluorescent green. Sections from each group were analyzed. Compared with the control group, cells in the CIA group showed a significant trend toward apoptosis (P < 0.001). The number of apoptotic cells in each treatment group was significantly decreased compared with the CIA group (P < 0.05, P < 0.01, or P < 0.001). The results further demonstrated that HUC-MSCs inhibited cell apoptosis, with the most pronounced inhibitory effect observed when combined with MTX.

[0403] 4. Summary and Discussion

[0404] The NF-κB signaling pathway is a classic inflammatory pathway that not only activates inflammatory effects but also promotes apoptosis. Studies have shown that inflammation and apoptosis jointly contribute to the development of RA. NF-κB p65 and p-p65 influence the onset and progression of inflammatory responses. p-p65 represents the activated state of p65. When the p-p65 / p65 ratio increases, activation of the NF-κB signaling pathway increases, promoting inflammation and apoptosis. When the p-p65 / p65 ratio decreases, inflammation and apoptosis are suppressed. Bax and Bcl-2 are involved in the apoptosis pathway. Bax promotes apoptosis, while Bcl-2 inhibits apoptosis. When the intracellular Bax / Bcl-2 ratio increases, a series of apoptosis-related signaling pathways are activated, prompting the cell to gradually initiate the apoptotic process and exhibit a clear propensity for apoptosis.

[0405] After hUC-MSCs treatment, Western blot, qRT-PCR, and TUNEL assays were used to detect inflammation and apoptosis under the activation of the NF-κB signaling pathway. The p-p65 / p65 and Bax / Bcl-2 ratios were significantly decreased, indicating that hUC-MSCs combined with MTX inhibited NF-κB signaling pathway activation, suppressed inflammatory expression, and prevented cells from abnormally entering the apoptotic program. In summary, hUC-MSCs combined with MTX inhibited NF-κB pathway activation by inhibiting p65 phosphorylation, regulating the Bax / Bcl-2 signaling pathway to achieve anti-inflammatory and anti-apoptotic effects, and had a regulatory effect on RA, providing a theoretical basis for the relevant mechanism of action of potential diagnostic and therapeutic targets for RA.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for treating rheumatoid arthritis, characterized in that: The pharmaceutical composition comprises mesenchymal stem cells and methotrexate.

2. The use according to claim 1, characterized in that The mesenchymal stem cells are human umbilical cord mesenchymal stem cells; the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

3. The use according to claim 1, characterized in that The rheumatoid arthritis is accompanied by joint swelling, arthritic cell infiltration, joint bone tissue destruction, articular cartilage structure destruction, synovial inflammation and / or abnormal cell apoptosis of synovial tissue.

4. The use according to claim 1, characterized in that The treatment is achieved by: 1) Reduce joint swelling; and / or 2) repair bone tissue; and / or 3) repairing cartilage tissue; and / or 4) alleviate arthritic cell infiltration; and / or 5) reducing serum levels of ACPA, IL-6, and TNF-α, and increasing serum levels of IL-10 and TGF-β; and / or 6) inhibiting cell apoptosis: reducing the p-p65 / p65 ratio and the Bax / Bcl-2 ratio; and / or 7) inhibiting synovial inflammation.

5. The use according to claim 1, characterized in that The pharmaceutical composition inhibits the activation of the NF-κB pathway by inhibiting p65 phosphorylation, and regulates the Bax / Bcl-2 signaling pathway to achieve anti-inflammatory and anti-apoptotic effects, thereby treating RA.

6. Use of a pharmaceutical composition in the preparation of a drug for alleviating RA inflammation, characterized in that: The pharmaceutical composition comprises mesenchymal stem cells and methotrexate.

7. Use of a pharmaceutical composition in the preparation of a medicament for reducing joint swelling, repairing joint bone tissue, repairing joint cartilage tissue and / or inhibiting synovial inflammation, characterized in that: The pharmaceutical composition comprises mesenchymal stem cells and methotrexate.

8. The use according to claim 7, characterized in that The pharmaceutical composition promotes the conversion of macrophages in the synovium from the M1 type that promotes inflammation to the M2 type that inhibits inflammation.

9. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that: The pharmaceutical composition comprises mesenchymal stem cells and methotrexate.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is a combination drug; or the pharmaceutical composition is prepared in the form of a compound drug into any pharmaceutically acceptable dosage form.

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