肾小球系膜细胞表达基质金属蛋白酶组织抑制物-1、-2及凝血酶调控的研究

Over-expression of TIMP-1, -2 in cultured human mesangial cells exposed to thrombin

目的探讨人肾小球系膜细胞基质金属蛋白酶组织抑制物（TIMP-1、－2）的表达及凝血酶对它们的调节作用，进一步阐明凝血酶促进肾小球损伤及硬化过程中的分子生物学机制。方法利用逆转录多聚酶链反应（RT－PCR）、Northern杂交和Western印迹等分子生物学方法，观察了凝血酶对体外培养的人肾小球系膜细胞表达TIMP－1、－2的诱导作用。结果在无血清RPMI1640培养12h后，RT－PCR、Northern和Western分析显示，系膜细胞可表达基础水平的TIMP-1、2mRNA及其蛋白质；凝血酶（0．5、15、4．5HIU／ml）可浓度依赖性地上调系膜细胞TIMP-1、-2mRNA的表达水平，Western印迹分析证明凝血酶还可以上调系膜细胞表达TIMP-1、－2蛋白水平；凝血酶的上述作用可被其特异性抑制物—水蛙素部分性阻断。结论正常人肾小球系膜细胞在基础状态下可以表达TIMP－1、－2，我们首次证实凝血酶在基因转录和蛋白质翻译水平促进系膜细胞表达TIMP-2，从而参与病理状态下肾小球内细胞外基质的异常代谢过程。

Objectives To further explore the underiying mechanism of thrombin in augmenting renal injury and the induction of thrombin in the expression of TIMP-1, -2 in cultured human mesangial cells. Methods Human mesangial cells incubated in serum-free RPMI 1640 were used and expression of TIMP-1, -2 in the cell exposed to thrombin were evaluated by means of RT-PCR, Northem hybridization, and Western blotting analysis. Results Cells exhibited low levels of basic expressions of TIMP-1, -2 mRNA and their protiens after 12 h incubation in serum-free RPMI 1640 medium. RT-PCR, Northern hybridization and Westem blotting showed that thrombin (0. 5, 1. 5, and 4. 5 HIU/ml respectively), in a concentraion-dependent manner, enhanced levels of TIMP-1, - 2 mRNA and their proteins in the cell treated for 12 h. All the results above were partially blocked by a pre-treatment of hirudin, a specific inhibitor of thrombin. Conclusions That thrombin can stimulate TIMP-1, -2 expression at both transcriptional and translational levels in human mesangial cells, which may contribute to evolution of glomerular injury and glomerulosclerosis augmented by thrombin in some renal diseases.