饰胶蛋白聚糖基因转染对大鼠肾系膜细胞转化生长因子βⅠ、βⅡ型受体表达的影响

Influence on the expression of type I and type II transforming growth factor-β receptors in cultured rat mesangial cells transfected by decorin gene

目的探讨饰胶蛋白聚糖(DCN)拮抗肾小球硬化进展的作用是否与其抑制系膜细胞(MsC)转化生长因子βⅠ、βⅡ型受体(TGF—βRⅠ、TGF—βRⅡ)表达有关。方法经外源性TGF—β1刺激培养的大鼠肾MsC,采用RT—PCR、Western印迹法检测其TGF—βRⅠ、TGF—βRⅡ的表达。用脂质体介导法将DCN基因转染MsC,经筛选和鉴定,用RT—PCR、Western印迹法和免疫荧光法分别观察DCN基因转染对TGF—βRⅠ、TGF—βRⅡ表达的影响。结果经外源性TGF—β1刺激的正常MsC,其TGF—βRⅠ、TGF—βRⅡ基因转录及蛋白表达均明显升高,且呈时间依赖性,于作用24h达高峰。与转染空载体的MsC(1P-1)相比,转染DCN基因的MsC克隆(3D-5,7D-1)的TGF-βRⅠmRNA表达分别为对照组的20%和32%,TCF-βRⅡmRNA表达分别为对照组的64%和59%;TGF—βRⅠ蛋白表达分别为对照组的34%和25%,TGF—βRⅡ蛋白表达分别为对照组的49%和57%。同时转染DCN基因也能阻止MsC对外源性TGF-β1刺激所致的两种受体表达升高作用。结论过表达DCN基因的MsC,其TGF-βRⅠ、TGF—βRⅡmRNA和蛋白表达水平均明显降低,这可能是DCN拮抗由TGF-β介导的肾小球硬化发生的重要机制之一。

Objective To explore whether the antagonistic effect of decorin (DCN)on progression of glomeruloselerosis is associated with the inhibition of the expression of type I and type II transforming growth factor-β receptors (TGF-βR I and TGF-βR II )in mesangial cells (MsC). Methods RT-RCR and Western blot analysis were used to detect the expression of TGF-βR I and TGF-βR II mRNA and their proteins on cultured rat MsC stimulated by exogenous TGF-β1. Lipofectin-mediated method was used to transfect DCN vector into MsC. After screening and identifying of transfected MsC, RT-PCR and Western blot analysis were adapted to detect the changes of TGF-βR I and TGF-βR II expression respectively. Results The expression of TGF-βR I and TGF-βR II mRNA and their proteins on normal MsC stimulated by exogenous TGF-β1 increased in time-dependent manner and reached the peak at 24th hour. Compared with normal and untransfected MsC (1P-1), the mRNA and protein expression of TGF-βR I and TGF-βR II on MsC (3D-5, 7D-1) transfecled by DCN gene decreased significantly, and DCN gene transfection could antagonize the increase of mRNA and protein expression of both receptors caused by exogenous TGF-β1. Conclusions The expression of both TGF-βR I and TGF-βR II decreases obviously in MsC overexpressing DCN gene, which may be one of the importan antagonistic mechanisms of decorin involved in the development of glomeruloselerosis mediated by TGF-β.