慢病毒介异的孕烷X受体sh-RNA稳定干扰HepG2细胞株的构建  

Establishment of a HepG2 cell line with stable PXR silencing by lentivirus-mediated RNA interference

作　　者：牛永东[1,2] 陈理明[3] 吴曼鹏 程訸 徐涵[1] 高分飞[1] 石刚刚[1] 

机构地区：[1]汕头大学医学院药理教研室,广东汕头515041 [2]上海市肿瘤研究所,癌基因及相关基因国家重点实验室,上海200032 [3]汕头大学医学院第一附属医院肿瘤科,广东汕头515041 [4]汕头市第二人民医院儿科,广东汕头515011

出　　处：《癌变．畸变．突变》2014年第3期209-212,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：中国博士后科学基金(2012M510200);广东省自然科学基金(S201 2010010955);广东省医学科研基金(A2013656);中央财政支持地方高校发展专项资金

摘　　要：目的:构建慢病毒介导的人孕烷X受体(hPXR)sh-RNA干扰载体及筛选肝癌细胞稳定干扰株。方法:针对hPXR的特异性序列,设计干扰序列,构建重组克隆。转染HEK293T细胞,筛选有效干扰片段。HEK293T包装干扰病毒并转染肝癌HepG2细胞株,200 mmol/L嘌呤霉素筛选2-3周,Western blot检测PXR表达。hPXR激动剂利福平处理PXR sh-RNA稳定干扰肝癌HepG2细胞株和Scramble对照细胞,抽提RNA并鉴定hPXR靶基因细胞色素P450 3A4(CYP3A4)的表达。结果:筛选出了PXR有效干扰片段,并获得了PXR信号通路有效敲低的HepG2慢病毒稳定干扰株。结论:成功构建了慢病毒介导的PXR sh-RNA稳定干扰HepG2细胞株。OBJECTIVE：To establish a HepG2 cell line with stable PXR silencing effect by a lentiviral vector carrying a short hairpin RNA （sh-RNA）. METHODS：Three double-stranded sh-RNA targeting the PXR gene were designed,synthesized and cloned into the psi-sH1 sh-RNA vector. The effective recombinant sh-RNA expression plasmid against PXR,which was selected of HEK293T cells with three double-stranded sh-RNA plasmids,was packaged into HEK293T cells and used to infect HepG2 cells after 200 mmol/L puromycin screening for 2-3 weeks. The expression level of PXR was detected by western blot,the CYP3A4 mRNA levels both in sh-RNA PXR HepG2 stable cells and scramble control HepG2 cells were detected by real-time PCR. RESULTS：A recombinant sh-RNA expression plasmid against PXR gene with effective interference was successfully selected and a HepG2 cell line stably transfected with the sh-RNA PXR was established. CONCLUSION：A HepG2 cell line stably transfected with the sh-RNA PXR was established.

关 键 词：人孕烷X受体 原发性肝细胞癌 慢病毒 RNA干扰 

分 类 号：R365[医药卫生—病理学]