【摘要】 目的研究转化生长因子(TGF)-β1对大鼠系膜细胞纤维蛋白溶酶(纤溶酶)系统的影响,并探讨反应性氧基(ROS)的作用。方法以体外培养的大鼠系膜细胞为对象,采用TGF-β1刺激12h,部分实验中培养的细胞在刺激前应用DL-buthionine-(S,R)-sulfoximine(BSO)预处理24h,或应用抗氧化剂N-acetylcysteine(NAC)预处理1h。应用荧光素法测定细胞ROS含量。系膜细胞tPA、uPA和PAI-1mRNA的表达采用RT-PCR测定。PAI-1蛋白表达应用ELISA检测。应用合成的荧光素酶底物及荧光扫描法分别测定纤溶酶、uPA、tPA活性。结果TGF-β1可显著增加细胞ROS含量。2ng/mlTGF-β1可显著上调PAI-1mRNA和蛋白的表达,分别为1.9倍(P<0.01)和1.5倍(P<0.05)。tPAmRNA的表达不受影响但uPAmRNA的表达较对照组下调52%(P<0.01)。TGF-β1可抑制纤溶酶和uPA活性,分别为30%(P<0.01)和23%(P<0.05),但对tPA的活性无显著影响。在BSO预处理组,TGF-β1对uPA、PAI-1mRNA表达和蛋白释放的影响得到显著增强;而NAC预处理可明显逆转由TGF-β1诱导的uPA、PAI-1mRNA表达的上调和蛋白表达增加以及活性的改变。结论TGF-β1可促使细胞ROS产生增加。ROS介导了由TGF-β1诱导的系膜细胞PAI-1mRNA和蛋白表达的上调以及uPAmRNA表达的下调和uPA、纤溶酶活性的下降。TGF-β1对系膜细更多还原

【Abstract】 Objective To explore the role of ROS in TGF β1 induced changes of plasmin system in rat mesangial cells. Methods Growth arrested and synchronized rat mesangial cells were stimulated by 2 ng/ml TGF β1 for 12 h. In some experiments cells were pretreated with BSO for 24 h or with antioxidant NAC for 1h. Intracellular ROS production was visualized using a fluorescent dye.The mRNA expression of tPA, uPA and PAI 1 was measured by RT PCR and PAI 1 protein secreted into media by ELISA assay.The activities of plasmin, uPA, tPA were determined using a synthetic fluorometric substrate. Results TGF β1 significantly increased intracellular ROS concentration. 2 ng/ml TGF β1 significantly upregulated PAI 1 mRNA and protein expression by 1 9 fold(P< 0 01)and 1 5 fold (P< 0 05),respectively. No significant change of tPA mRNA expression was observed with uPA mRNA expression significantly decreasing to 52%of control(P< 0.01). TGF β1 suppressed plasmin and uPA activities by 30%(P< 0 01) and 23%(P< 0 05) respectively, but tPA activity was unaffected. TGF β1 induced changes in uPA, PAI 1 mRNA expression and protein secretion were exaggerated by BSO. Antioxidant NAC effectively reversed above changes and their activities.Conclusions TGF β1 can significantly increase intracellular ROS concentration in mesangial cells. TGF β1 induced upregulation of PAI 1 mRNA and protein expression,downregulation of uPA mRNA expression, and suppression of uPA and plasmin activities in mesangial cells are mediated by ROS. Plasmin activity in mesangial cells by TGF β1 acts mainly throngh the suppressing of uPA expression and activity.更多还原