siRNA下调Smad3基因后对多肽N-乙酰氨基半乳糖转移酶表达的影响  

Knock-down of Smad3 Gene by siRNA and Its Effect on Expression of Polypeptide:N-acetyl-galactosaminyl Transferase

作　　者：胡从莉[1,2] 王泽荣[1,2] 孙斌[1,2] 徐正荣[1,2] 吴士良[1,2] 

机构地区：[1]苏州大学医学部生物化学与分子生物学系,江苏苏州215123 [2]苏州大学生化工程研究所,江苏苏州215123

出　　处：《苏州大学学报（医学版）》2009年第5期830-833,I0001,共5页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(30670462)

摘　　要：目的以Smad3基因为研究对象,筛选出该基因的有效siRNA干扰载体,并探讨下调表达Smad3基因对多肽N-乙酰氨基半乳糖转移酶(ppGalNAcTs)家族mRNA水平表达的影响。方法将事先构建的3个带有荧光标记的干扰载体分别转染人肝星状细胞株(HSC)中,通过RT-PCR、Western blot方法分别从mRNA水平及蛋白水平检测干扰效率。并通过转染有效的Smad3基因的siRNA表达载体,检测下调Smad3以后对糖基转移酶ppGalNAcTs家族mRNA表达的影响情况。结果成功筛选到Smad3基因的有效siRNA干扰载体,检测出转染了Smad3的siRNA表达载体的肝星状细胞中Smad3的mRNA和蛋白表达都受到了抑制,而糖基转移酶ppGalNAcTs的mRNA表达也随之发生了一些变化。结论成功筛选到一个Smad3基因的siRNA干扰载体,继而可转染人肝星状细胞,并抑制其中Smad3蛋白的表达,为研究siRNA方法在肝纤维化中的治疗作用提供了实验基础和理论支持。Objective To screen the optimal RNAi-vector of Smad3,and to study the mRNA expression of ppGalNAcTs in hepatic stellate cells（HSCs） transfected with the Smad3 RNAi-vector. Methods RT-PCR and Western blot methods were used to screen a optimal Smad3 RNAi-vector,and RT-PCR to detect the mRNA expression of ppGalNAcTs after HSC transfected with the optimal Smad3 RNAi-vector. Results An optimal RNAi-vector of Smad3 was obtained and the mRNA expression of ppGalNAcTs was slightly down regulated after the knock-down of Smad3 gene by the optimal siRNA. Conclusion The knock-down model of Smad3 gene is successfully constructed,and this provides a support for the RNAi method in the treatment of hepatic fibrosis.

关 键 词：SMAD3基因 SIRNA 肝纤维化 转化生长因子-Β 多肽N-乙酰氨基半乳糖转移酶 

分 类 号：R575.02[医药卫生—消化系统]