siRNA阻断肌成纤维细胞内源性Smad3表达的研究  

Inhibition of endogenous Smad3 expression in C2C12 cells by siRNA

作　　者：邓长柏[1] 杨作成[2] 

机构地区：[1]中南大学湘雅三医院儿科,湖南长沙410013 [2]南方医科大学南方医院儿科,广东广州510515

出　　处：《临床儿科杂志》2007年第10期815-817,821,共4页Journal of Clinical Pediatrics

基　　金：湖南省卫生厅科研基金(B2005-072);教育部留学回国启动基金(2006-311)

摘　　要：目的观察Smad3基因的RNA干扰(RNAi)对转化生长因子β1(TGF-β1)诱导的小鼠肌成纤维细胞(C2C12)的Smad3表达。方法实验分为实验组、内对照组和空白对照组。用不同浓度TGF-β1干预C2C12细胞,利用体外转录合成的siRNA-Smad3,以阻断Smad3基因表达。用Westernblot检测Smad3和磷酸化的Smad3。结果5ng/mlTGF-β1干预C2C12细胞,促进Smad3磷酸化。0.5h开始诱导Smad3磷酸化,5h达高峰。TGF-β1干预C2C12细胞,诱导Smad3磷酸化呈剂量依赖性。siRNA-Smad3转染C2C12细胞24h,Smad3表达逐渐被抑制到无表达。siRNA-Smad3转染C2C12细胞24h后,加5ng/mlTGF-β1干预,Smad3无表达。结论TGF-β1促进C2C12细胞Smad3磷酸化呈剂量与时间依赖性;siRNA阻断C2C12细胞Smad3表达与Smad3磷酸化。Objectives To determine the effects of RNA interference inhibition on Smad3 expression in C2C12 cells induced by TGF-β1. Methods C2C12 cells were divided into three groups： experimental group, internal control group and blank control group. Western blotting was used to detect the protein expression of Smad3 and phosphorylated Smad3 in C2C12 cells induced by TGF-β1 at different concentrations and times. Results TGF-β1 （5 ng/ml） was used to induce C2C12 cells and stimulate phosphorylation of Smad3. The phosphorylation of Smad3 began at 0.5 h after TGF- β1 was added and reached the peak at 5 h. C2C12 cells were treated with different concentrations of TGF-β1 （0, 1, 5, and 10 ng/ml） for 5 h. The phosphorylation of Smad3 increased in a dose-dependent manner. In C2C12 cells transfected with siRNA-Smad3 for 24 h, Smad3 expression decreased gradually and the expression was blocked completely at the highest dose. Such phenomenon was not observed in the internal and blank control groups. C2C12 cells transfected with 200 pmol/L of siRNA-Smad3 were treated with 5 ng/ml of TGF-β1 for 5 h. No Smad3 expression was observed in these cells. While the expression of endogenous and phosphorylated Smad3 was found in the internal control group. Conclusions TGF-β1 could enhance the phosphorylation of Smad3 in C2C12 cells in a dose and time dependent manner, siRNA can inhibit Smad3 expression and phosphorylation in C2C12 cells.

关 键 词：SMAD3 短干扰RNA 转化生长因子Β1 成纤维细胞 

分 类 号：R363[医药卫生—病理学]