人Smad3的shRNA表达载体构建和鉴定  

Construction and characterization of human Smad3 shRNA expression vector

作　　者：谢桥生[1] 雷小英[2] 王立锋[2] 孟艳玲[1] 王芳[2] 薛茜[1] 温伟红[1] 杨安钢[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,陕西西安710033 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2008年第2期104-106,共3页Journal of the Fourth Military Medical University

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0459)

摘　　要：目的:构建人Smad3的shRNA表达载体,运用RNAi下调Smad3基因在肿瘤细胞系中的表达,观察对肿瘤细胞生长的影响.方法:化学合成针对人Smad3基因的dsRNA,瞬时转染入Hela细胞,检测RNA干扰效果,筛选有效干扰靶位点;设计并合成针对人Smad3基因的siRNA寡核苷酸链,经退火形成双链后克隆入质粒载体pSilencerTM3.1-H1hygro,转染入Hela细胞,用hygromycinB筛选稳定单克隆细胞株,对细胞株行RT-PCR和WesternBlot检测,观察siRNA对靶基因的抑制效果,进一步研究下调目的分子表达水平以后肿瘤细胞生长的变化.结果:瞬时转染筛选到了有效的RNA干扰靶位点,构建载体后,建立稳定转染的单克隆细胞株,Smad3mRNA和蛋白质水平均显著下调,导致肿瘤细胞生长抑制.结论:成功构建了针对人Smad3高效、特异、作用持久的shRNA表达载体,转染细胞后可下调Smad3的表达,为进一步研究Smad3的功能和肿瘤的基因治疗奠定了基础.AIM： To construct human Smad3 shRNA expression vector, and to observe the effect of Smad3 gene knocking down on tumour cell proliferation by depressing human Smad3 gene expression with RNA interference in tumour cell line. METHODS： Double-stranded RNA targeting human Smad3 gene was synthesized chemically, to transfect Hela cell line transiently and choose the efficacious siRNA sequence. An effective sequence was designed and cloned into shRNAs expression plasmid vector pSilencerTM3. 1-HI hygro. The constructed plasmid was stably transfected into Hela cells, and stable monoclone was selected within hygromycin B media. RT-PCR and Western Blot were performed to monitor the expression and interference efficiency of siRNAs against Smad3, and then to observe the consequent effect on Hela cell proliferation. RESULTS： The effective siRNA sequence was screened by transient transfection. The vector expressing human Smad3 shRNA was successfully constructed and transfected stably into Hela cells. The growth of the monoclone cells was inhibited evidently by the down-regulation of Smad3 both at mRNA and protein levels, as detected by RT-PCR and Western Blot. CONCLUSION： Plasmid vector expressing small hairpin RNA against Smad3 has been successfully produced and it can down-regulate Smad3 expression after transfected into cells,which will facilitate further studies of Smad3 function and its application in tumour gene therapy.

关 键 词：SMAD3 RNA干扰 遗传载体 肿瘤生长抑制 

分 类 号：R737.33[医药卫生—肿瘤] Q782[医药卫生—临床医学]