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目的 比较类风湿关节炎 (rheumatoidarthritis,RA)与骨关节炎 (osteoarthritis,OA)成纤维样滑膜细胞 (fibroblast likesynoviocytes,FLS)蛋白酪氨酸磷酸化状态的差异 ;探讨RAFLS中 ,蛋白酪氨酸激酶 (proteintyrosinekinase,PTK)在IL 1β和TNF α作用下有丝分裂原活化蛋白激酶 (mito genactivatedproteinkinase ,MAPKs)活化中的作用。 方法 滑膜细胞原代培养 ,流式细胞仪鉴定滑膜细胞型别 ,提取蛋白进行SDS PAGE单相电泳和ICE PAGE双相电泳分离后 ,用Westernblots检测FLS蛋白磷酸化的状态。结果 滑膜细胞培养至第三代时 ,CD3、CD14、CD2 0、CD11b、vonWillebrandfactor阳性细胞比例小于 1% ;RAFLS蛋白酪氨酸磷酸化程度是OAFLS的 4～ 6倍 ;IL 1β和TNF α可以瞬时引起RAFLS蛋白酪氨酸磷酸化程度增加 ,并在短时间内激活MAPKs(ERK2 ,JNK2 和p38为主 ) :genistein对ERK2 活化抑制作用显著 ,而对于JNK2 、p38,活化的抑制则较弱。 结论 原代培养的滑膜细胞当传代至第三代时即可以获得型别均一的成纤维样滑膜细胞 ;RAFLS蛋白酪氨酸磷酸化状态较OAFLS明显增高 ;IL 1β和TNF α可以瞬时引起RAFLS蛋白酪氨酸磷酸化程度增加 ,激活MAPKs通路 (EPK2 、JNK2 、p38) ,两种因子诱导MAPKs活化过程中PTK依赖途径和PTK非
Objective To compare the tyrosine phosphorylation status between rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast likeynoviocytes (FLS), and to explore the role of protein tyrosine Activation of mitogen-activated protein kinases (MAPKs) by IL-1β and TNFα by protein tyrosine kinase (PTK). Methods Synovial cells were cultured in primary culture. The synoviocyte types were identified by flow cytometry. The proteins were separated by SDS PAGE and ICE PAGE. The phosphorylation of FLS was detected by Western blot. Results The percentage of CD3, CD14, CD20, CD11b and von Willebrand factor positive cells in synovial cells was less than 1% at the third passage. The tyrosine phosphorylation of RAFLS protein was 4-6 times higher than that of OAFLS. IL 1β and TNFα The transient increase of tyrosine phosphorylation of RAFLS protein and the activation of MAPKs in short time (ERK2, JNK2 and p38): genistein significantly inhibited the activation of ERK2 and inhibited the activation of JNK2 and p38. CONCLUSIONS: Primary cultured synovial cells can form homogeneous fibroblast-like synoviocytes when passaged to the third generation. The tyrosine phosphorylation status of RAFLS protein is significantly higher than that of OAFLS. IL-1β and TNFα can be transiently induced RAFLS protein tyrosine phosphorylation increased activation of MAPKs pathway (EPK2, JNK2, p38), two factors in the activation of MAPKs PTK-dependent pathway and PTK-dependent