【摘要】 目的　探讨转化生长因子 β1(TGF β1) /Smad信号通路对大鼠肾系膜细胞 (MsC)基质金属蛋白酶 2 (MMP 2 )及其组织抑制因子 2 (TIMP 2 )蛋白表达和酶活性的影响。方法　采用脂质体介导法 ,分别将Smad 2 3 7基因瞬时转染体外培养的大鼠MsC ,并用免疫荧光 逆转录 聚合酶链反应 (RT PCR)和Western印迹法检测其转染成功与否 ;再分别用Western印迹 酶谱法或反式酶谱法 ,观察转基因MsC及其在TGF β1作用下MMP 2和TIMP 2蛋白表达和酶活性的改变。 结果　转染Smad 2基因的MsC ,MMP 2和TIMP 2蛋白表达和酶活性均略有升高 ,蛋白表达分别增强 1 4倍和 1 1倍 ;酶活性分别提高 1 4倍和 1 3倍 ,经TGF β1作用后有明显升高 ,蛋白表达分别增加 2 8倍和 3 0倍 (P <0 0 1) ;酶活性分别提高 3 0倍和 1 7倍 (P <0 0 1) ;转染Smad 3基因的MsC ,TIMP 2蛋白表达和酶活性略有升高 ,蛋白表达增强 1 2倍 ,酶活性提高 1 2倍 ,TGF β1作用后升高更为明显 ,蛋白表达增强 2 9倍 ,酶活性提高 1 8倍 ,而MMP 2蛋白表达和酶活性均无明显变化 ;转染Smad 7基因的MsC ,MMP 2和TIMP 2蛋白表达和酶活性均略有下降 ,蛋白表达均降低 1 2倍 ,酶活性均下降 1 2倍 ,在TGF β1作用下更为明显 ,蛋白表达分别?更多还原

【Abstract】 Objective To explore the effect of transforming growth factor (TGF) β1/ Smad signaling pathway on the expression and enzymatic activity of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in cultured rat mesangial cells (MsC). Methods Lipofectin method was used to transfect Smad 2, Smad 3 and Smad 7 vectors into MsC; and immunofluorescence, RT-PCR and Western blot analysis were used to detect their transfection efficiency. The expression and enzymatic activity of MMP-2 and TIMP-2 were determined by Western blot, zymography or reverse zymography assay. Results MsC transfected with Smad 2 gene showed slightly increased expression and enzymatic activity of both MMP-2 and TIMP-2, which was more obvious upon stimulation by TGF-β1. MsC transfected with Smad 3 gene showed a slight upregulation of TIMP-2 expression and its enzymatic activity, which was enhanced after TGF-β1 stimulation. There was however no change in MMP-2 expression and its enzymatic activity. On the other hand, MsC transfected with Smad 7 gene showed a decrease in MMP-2 and TIMP-2 expression and enzymatic activity, which was especially obvious after stimulation by TGF-β1. Conclusions TGF-β1/Smad signaling pathway may play an important role in the pathogenesis of glomerulosclerosis, probably via MMP-2 and TIMP-2 expression and the associated enzymatic activity.更多还原