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蛋白酪氨酸激酶在IL |
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n passage 3,the positive cells′ proportion of CD3,CD14,CD11b,CD20,and von Willebrand factor less than 1% respectively.The extent of protein tyrosine phosphorylation in RA FLS is 4～6 times higher than that of OA FLS.IL-1β and TNF-α transiently increased protein tyrosine phosphorylation,and activated MAPKs cascades (mainly ERK2,JNK2,and p38) in RA FLS.The activation of ERK2 induced by the two cytokines was inhibited obviously by genistein,but the inhibitory role on that of JNK2 and p38 was relatively weak.Conclusion Synoviocytes of passage 3 are homogenous population of fibroblast-like synoviocytes;tyrosine phosphorylation status in RA FLS increases much higher than that of OA FLS.During signal transductionf IL-1β and TNF-α in RA FLS,tyrosine phosphorylation is increased transiently;MAPKs cascades are activated in a few minutes (mainly ERK2,JNK2 and p38),PTK has a role in activation of ERK,but has weak effect on that of JNK and p38.
【Key words】 Arthritis; Synovial membrane; Fibroblasts; Protein tyrosine kinase; Mitogen activated protein kinase; Interleukin-1; Tumor necrosis factor
类风湿关节炎(rheumatoid arthritis，RA)是一种慢性系统性疾病，它的病情进展中综合体现了炎症、自身免疫、滑膜组织增生三种病理生理过程。异常增生的滑膜组织类似于局限性侵袭生长的肿瘤，对关节软骨、骨造成进行性破坏，最终导致关节畸形和不同程度的功能障碍。最近研究发现，RA成纤维样滑膜细胞(fibroblast-like synoviocyte，FLS)具有转化细胞特征，而在骨关节炎中未发现此转化现象的存在。这种转化特性可能是RA疾病发生的早期特征［1］。蛋白酪氨酸激酶在细胞转化和生物信号转导中发挥关键作用［2］。本研究通过比较RA和OA两种不同发病机制的关节病变中成纤维样滑膜细胞酪氨酸磷酸化状态的差异，探讨RA成纤维样滑膜细胞中酪氨酸激酶活性的改变；应用IL-1β、TNF-α作用于RA FLS， 观察两种细胞因子对有丝分裂原活化蛋白激酶(mitogen activated protein kinase,MAPKs)的激活情况，并应用genistein(特异性酪氨酸激酶抑制剂)，探讨酪氨酸激酶在上述因子诱导RA FLS MAPKs亚家族成员活化的信号转导过程中发挥的作用。
1 资料与方法
1.1 滑膜细胞的培养：滑膜均取材于北京医科大学人民医院骨关节病诊疗研究中心行膝关节置换术或滑膜切除术的病人，其中RA 20例，OA 20例，所有病人均符合国际诊断标准［3］。术中取出滑膜组织，无菌条件下剪碎，1.0 mg/ml Ⅰ型胶原酶(GIBCO-BRL) 37 ℃消化4 h，离心收集细胞，加入20% FBS-DMEM (GIBCO-BRL)培养液中，置于37 ℃、5% CO2孵箱中令其贴壁生长，24 h后PBS冲洗除去悬浮细胞，待细胞贴壁生长至85%汇满时，胰蛋白酶-EDTA消化进行1∶3传代。本实验采用均为3～5代滑膜细胞。
1.2 滑膜细胞类型鉴定：分别取1～3代滑膜细胞，用0.125 mmol上一页 [1] [2] [3] 下一页
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