Effects Of Four Drugs On The Levels Of IL-1β, MMP-13,PGE2Secreted By OA Synovial Fibroblast

First part:Isolation and culture of synovial fibroblastObject:To collect OA synovial tissue and normal synovial tissue, establish the synovial fibroblasts in vitro culture system.Methods:Collect the synovial membrane of OA patients and healthy persons. Use enzyme digestion method to get the original generation of synovial cells. Use flow cytometry to analyse expression of CD90+, CD3+and CD14+of cell. Use MTT method to measure the growth curve of the two kinds of synovial cell.Results:The expression of cell surface is CD3+2.53%, CD14+3.75%, CD90+96.11%, consistent with synovial fibroblasts. There are no different in proliferation rate between the normal and OA synovial fibroblast.Conclusion:The method of this experiment can get cultured synovial fibroblast successfully, the purity of synovial fibroblasts is more than96%. There are no different in proliferation rate between the normal and OA synovial fibroblast. Second part:The effection of four drugs on the level of IL-1β, MMP-13,PGE2secreted by OA synovial fibroblastObject:To explore the specific effects of four drugs to OA synovial fibroblast, and experiment could the various drug two two mixture can achieve a better effect. At the same time make a feasibility analysis for celecoxib intra-articular injection.Methods:Use dexamethasone, celecoxib, rhein and sodium hyaluronate to influence in synovial fibroblasts for24h, then use MTT method to measure the survival fraction value of synovial fibroblasts. Use sandwich ELISA method to measure the IL-1β, MMP-13, PGE2concentration of the supernatants.Results:The OA synovial cells in vitro culture are higher than normal synovial cells in the level of the IL-1β, MMP-13, PGE2; Rhein can reduce the IL-1β and MMP-13levels, but can not reduce the level of PGE2; celecoxib can reduced the secretion level of IL-1β and PGE2, but can increase the secretion of MMP-13. Dexamethasone can reduce the secretion level of IL-1β, PGE2, MMP-13. Rhein and dexamethasone have synergistic effect in reducing the IL-1level.Conclusion:1. There are synovitis disease. in the process of OA.2Rhein can reduce the level of IL-1β, MMP-13, but does not reduce the level of PEG2.3Celecoxib can reduce the level of IL-1β, PGE2, but can increase the levels of MMP-13. 4Dxamethasone can reduce the IL-1β, MMP-13and PGE2levels. Dexamethasone and rhein can synergistically reduces IL-1β3.5The co-effection of celecoxib and rhein can reduce IL-1β, MMP-13and PGE2levels. The third part:Effects of four drugs on the levels of IL-1β, MMP-13,PGE2secreted by OA synovial fibroblast influenced by LPSObjective:To explore the specific effects of various drugs to OA synovial cell with inflammation,and experiment could the various drug two two mixture can achieve a better effect. At the same time make a feasibility analysis for celecoxib intra-articular injection.Method:Use bacterial lipopolysaccharide (LPS) induce the inflammatory response of invitro cultured synovial cells, leading to establish the model of OA patients with synovitis model.Use LPS10mg/L and different concentrations of dexamethasone sodium phosphate, celecoxib, rhein, sodium hyaluronate and two two drug mixture to influence invitro cultured OA synovial cells for24h, then use MTT method to measure the survival fraction value of Synovial cells. Use sandwich ELISA method to measure the IL-1β, MMP-13, PGE2concentration of the supernatants. Results:The OA synovial cells in vitro culture are higher than normal synovial cells in the level of the IL-1β, MMP-13, PGE2; Rhein can reduce the IL-1β and MMP-13levels, but can not reduce the level of PGE2; celecoxib can reduced the secretion level of IL-1β and PGE2, but can increase the secretion of MMP-13. Dexamethasone can reduce the secretion level of IL-1β, PGE2, MMP-13. Rhein and dexamethasone have synergistic effect in reducing the IL-1level. Sodium hyaluronate can reduce the secretion level of IL-1β, PGE2, MMP-13.Conclusion:1Bacterial lipopolysaccharide can induce the inflammatory reaction in synovial cells.2Sodium hyaluronate can reduce the proinflammatory effects. Of LPS.3Rhein can reduce the level of IL-1β, MMP-13, but does not reduce the level of PEG2.4Celecoxib can reduce the level of IL-1p, PGE2, but can increase the levels of MMP-13.5Dxamethasone can reduce the IL-1β, MMP-13and PGE2levels. Dexamethasone and rhein can synergistically reduces IL-1β.6The co-effection of celecoxib and rhein can reduce IL-1β, MMP-13and PGE2levels.