| Objective:To investigate the therapeutic effects of human amnion-derived mesenchymal stem cells(hAMSCs)and tumor necrosis factor receptor antibody(tumor necrosis factor receptor inhibitor,iTNF)drugs on adjuvant rheumatoid arthritis(RA)in rats,as well as the mechanism of regulating the relevant factors in the inflammatory axis of IL-23/IL-17.It will provide experimental basis for improving the therapeutic effect of RA and finding new therapeutic targets.Methods:hAMSCs were isolated and cultured by using two-step enzymatic digestion combined with differential attachment method.Then were identified by flow cytometry and immunocytochemistry.The differentiation ability of hAMSCs into chondroblasts was confirmed by alcian blue and toluidine blue staining;Intra-articular injection of complete freund's adjuvant adjuvant to establish RA rat's model.hAMSCs were transplantated via sublingual intravenous injection,Weight and AI were measureed dynamically,Left ankle joint volume were measured by volume method,In vivo optical imaging of small animals was used to trace the colonization and distribution of hAMSCs.MRI were used to observe the morphology of the affected joint,cartilage and bone defects;HE and immunohistochemical staining were performed to observe the histopathologic changes and the expression of MMP-3 and MIP-3? in ankle joint tissue.Inflammatory cytokines in serum and joint fluid were detected by ELISA.Results:(1)P3 generation hAMSCs showed adhered growth,It expressed vimentin in cytoplasm and also highly expressed CD90,CD105,CD73 and CD44,but negatively expressed CD34,CD45,CD11 b,CD19 and HLA-DR.hAMSCs can be induced to differentiate into chondroblasts.(2)Compared with the normal group,the weight of each group increased slowly,the joint redness and swelling of the two treatment groups decreased,and the AI decreased,but on the 11 th day,the iTNF-? group reduced the joint volume,and the hAMSCs group significantly reduced the joint volume on the 15 th day.(3)In vivo tracer showed that XenoLight DiR labeled hAMSCs were transplanted into the damaged ankle joint on both day 7 and day 21 after transplantation.(4)MRI imaging showed that the normal group had clear ankle joint tissue structure,uniform signal in all layers,normal joint space,and no defect in articular cartilage and bone.In the model group,a wide range of high-signal changes occurred in the joint cavity,resulting in defects and deformations of the joint bone surface and enlargement of the joint space.In the iTNF-? group and hAMSCs group,the high signal in the joint cavity was significantly decreased,the gap was reduced,and the destruction of articular cartilage and bone was reduced.(5)Pathological examination showed that the model group had synovial hyperplasia,vasospasm and inflammatory cell infiltration compared with the normal group with normal structure.The cartilage surface was thinned,the transition layer and calcification layer were blurred,and the iTNF-? group was observed.Synovial hyperplasia and inflammatory cell infiltration were alleviated in the hAMSCs group,and cartilage and bone erosion were slowed down.(6)Immunohistochemistry of ankle joint MMP-3 showed that there was very little MMP-3 expression in the cartilage surface in the normal group,and high MMP-3 protein expression in the cartilage,bone and bone marrow cavity in the model group.MMP-3 was significantly decreased in the iTNF-? group and the hAMSCs group.(7)In the normal group,the synovial tissue of the normal group had low or no expression of MIP-3? protein,and the expression of MIP-3? protein in the model group increased,while the expression levels in the iTNF-? group and the hAMSCs group decreased significantly.(8)Compared with the normal group,the levels of inflammatory factors such as TNF-?,IL-1?,IL-17 A,IL-23,IFN-? and IL-6 in the serum and synovial fluid of the model group were significantly increased.the levels of pro-inflammatory factors were significantly decreased in the iTNF-? group and hAMSCs group,and the anti-inflammatory factors TGF-?1 and IL-10 were increased.However,the ability of iTNF-? group to reduce IL-23 was stronger than that of hAMSCs group,while the level of IL-10 in hAMSCs group was higher than that of iTNF-? group.Conclusion: hAMSCs has a significant effect on adjuvant induced RA in rats,and its effect is comparable to that of iTNF-?,but the effect of iTNF-? is faster than that of hAMSCs vein transplantation.iTNF-? down-regulates IL-23 more strongly than hAMSCs,but hAMSCs up-regulates IL-10 more strongly.The cartilage differentiation ability of hAMSCs and its regulation of IL-17 A and IL-10 can be distinguished from the main molecular immune mechanism of iTNF-?. |
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