人甲胎蛋白基因启动子驱动siRNA表达载体的构建及其在HepG2细胞中RNA效应的初步鉴定

目的：探索基于人甲胎蛋白基因启动子构建的siRNA表达载体介导目的基因RNA干扰的可行性，为实现肝癌细胞靶向RNAi治疗作一初步研究。方法：PCR法扩增获得人甲胎蛋白基因启动子并插入pSilencer1．0-U6中以置换其U6启动子；依据人hIRH-1基因RNAi靶位点设计出siRNA模板DNA,体外合成相应寡核苷酸片段后经退火形成短双链。再克隆构建成靶向人hLRH-1基因siRNA表达载体；脂质体转染法将上述重组质粒转入HepG2细胞中，实时定量RT—PCR法初步分析所建系统在AFP表达细胞中触发靶基因hIRH-1的RNAi效应，以及hIRH-1调控下游靶基因的表达改变。结果：获得人甲胎蛋白基因启动子驱动的siRNA表达载体pSWFPo以人hLRH-1为报告基因，本研究发现所建的RNAi载体系统可在表达AFP的细胞HepG2中有效诱导靶基因表达下调，抑制率约达85％；同时引致hLRH-1调控下游靶基因CydinEl与Oct4的表达相应下调。结论：人甲胎蛋白基因启动子驱动的siRNA表达载体可在表达AFP的细胞HepG2中有效介导靶基因的RNAi效应，为临床肝癌细胞靶向的基因特异性RNAi治疗提供了初步的实验依据。

CONSTRUCTION OF SIRNA EXPRESSION VECTOR DRIVING BY PROMOTER OF HUMAN ALPHAFETOPROTEIN GENE AND THE PRELIMINARY IDENTIFICATION OF ITS RNAI EFFECT IN HEPG2

Objective:To explore the possibility of RNA interference of target gene medieted by a siRNA expression vector driving by human alpha-fetoprotein gene promoter,thus offer a primary study for fulfilling the targeting RNAi therapy of hepato- cellular carcinoma.Method:The promoter of human AFP gene was amplified with PCR and inserted into pSilencer1.0-U6 to re- place its U6 promoter.DNA template of siRNA targeting human hLRH-1 was designed according to a formerly selected RNAi site.After synthesized in vitro an...