Smad3基因siRNA表达载体的构建及沉默效应  

Construction of a siRNA vector targeting Smad3 gene and gene-silencing effect

作　　者：王爱丽 吴志宏 徐顺 顾耀辉 黄静 陈波 贾卿 

机构地区：[1]Department of Burn and Plastic Surgery, Shanghai Seventh People＇s Hospital, Shanghai 200137, China

出　　处：《中国组织工程研究与临床康复》2011年第28期5285-5289,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：上海市浦东新区社会发展局青年基金资助项目:PW2005B-4,课题名称:siRNA抑制Smad3基因治疗增生性瘢痕的实验研究;上海市重点专科建设基金资助项目:05Ⅱ014,课题名称:烧伤瘢痕防治重点专科建设~~

摘　　要：背景:研究提示抑制Smad3可望抑制纤维增生和胶原的大量产生。目的:构建针对Smad3基因的siRNA表达载体,观察RNA干扰对增生性瘢痕成纤维细胞Smad3基因表达的抑制作用。方法:根据siRNA设计原则,设计3条针对Smad3基因的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后与载体pRNAT-U6连接,然后进行酶切鉴定和DNA序列测定。用脂质体包裹转染增生性瘢痕成纤维细胞,荧光定量PCR和Western blot方法检测Smad3基因的表达情况。结果与结论:测序分析结果显示:克隆入pRNAT-U6载体的针对Smad3基因的siRNA的双链寡核苷酸片段插入正确;荧光定量PCR和Western blot检测显示:转染的增生性瘢痕成纤维细胞Smad3基因的表达水平明显降低,尤以AGA CAG ACT GTG ACC AGT A(1156)为靶序列的siRNA沉默作用最强,抑制效率于转染后48h可达45%。证实实验构建的沉默增生性瘢痕成纤维细胞Smad3基因表达的siRNA载体获得成功。BACKGROUND：Previous studies demonstrated that inhibition of Smad3 can suppress fibroplasias or collagen over expression. OBJECTIVE：To construct an expression vector of siRNA targeting human Smad3 gene and observe its gene-silencing effect in hypertrophic scar （HS） fibroblasts METHODS：The siRNA sequences targeting Smad3 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6- Smad3 was transfected into human HS fibroblasts. Smad3 expressions in mRNA and protein levels were examined by real-time-PCR and Western blotting. RESULTS AND CONCLUSION：The result of recombinant sequence was the same as aim sequence. Real-time RCR and Western blot showed that, compared to blank, the mRNA and protein levels of Smad3 were significantly decreased. Thereinto, the most effective siRNA sequence was AGA CAG ACT GTG ACC AGT A （1156）. The gene-silent efficiency was 45%. Smad3-siRNA recombinant was constructed successfully and can be transfected into HS fibroblasts to inhibit the expression of Smad3 gene.

关 键 词：小分子RNA RNA干扰 SMAD3 表达载体 构建 基因沉默 

分 类 号：R737.33[医药卫生—肿瘤]