Application of human synovial membrane fibroblasts to treating rheumatoid arthritis
A fibroblast, rheumatoid technology, applied in medical preparations containing active ingredients, anti-inflammatory agents, non-central analgesics, etc. , improve the effect of occurrence and progression
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Embodiment 1
[0026] Example 1: Isolation and culture of fibroblasts derived from human synovial tissue
[0027] 1. Method
[0028] (1) Take the knee joint synovial tissue of osteoarthritis patients under aseptic conditions, and obtain P3 generation synovial fibroblasts through shredding, digestion, filtration, culture, and passage;
[0029] (2) Morphological observation of P3 generation synovial fibroblasts under a microscope;
[0030] (3) P3 generation fibroblasts were cultured in adipogenic differentiation medium and osteogenic differentiation medium for 21 days, stained with Oil Red O and Alizarin Red respectively, and detected and analyzed the multidirectional differentiation potential;
[0031] (4) The method of flow cytometry is used to detect the surface antigen molecules of P3 generation fibroblasts.
[0032] 2. Results
[0033] Biological properties of synovial fibroblasts such as figure 1 shown, where figure 1 (a) shows the fibroblast-like adherent growth of synovial fibrobl...
Embodiment 2
[0034] Example 2: Therapeutic effect of fibroblasts derived from human synovium injected into the knee joint cavity on CIA mice
[0035] 1. Method
[0036] (1) Establishment of type II collagen-induced arthritis (CIA) mouse model: All mice in the model group were subcutaneously injected with 100 μg of bovine type II collagen at the base of the tail on d0 and d21;
[0037] (2) Treat CIA mice with fibroblasts derived from human synovium injected into the right knee joint cavity: use 10 μl containing 1×10 6 PBS of fibroblasts was injected into the right knee joint cavity of CIA mice on d28, d32 and d38 days after the first immunization; 10 μl PBS was injected into the right knee joint cavity of the control group;
[0038] (3) Evaluation of CIA arthritis: score the arthritis of the mice in the model group every other day after the second immunization. The scoring method was double-blind, and the scoring criteria were: 0, normal; 1, redness involving one finger joint; 2, ≥ 2 fing...
Embodiment 3
[0041] Example 3: Detection of mRNA expression levels of cytokines in synovial tissue
[0042] On the 70th day, the mice in the control group and the fibroblast treatment group of Example 2 were sacrificed, the patella and adjacent synovial tissues were separated, and the mRNA levels of cytokines in the synovial tissues were detected by Real time-PCR.
[0043] The result is as image 3 As shown, it can be seen from the results that human synovium-derived fibroblasts can reduce tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) inflammatory factor IL-17A and transforming growth factor (TGF -β) secretion, and up-regulate the levels of immune regulatory molecules IL-10 and IL-4. It can be seen that the fibroblasts derived from human synovium have immunoregulatory effect and tissue repair ability, and will have broad application prospects in the field of rheumatoid arthritis treatment.
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