DJ-1 shRNA表达载体的构建及其稳定转染人肝癌细胞HepG2细胞株的建立  

Construction of shRNA expression vector targeting D J-1 and screening of stably transfected HepG2 cells

作　　者：刘顺芳[1] 刘谨文[1] 易继林[1] 杨志芳[1] 殷茜[1] 李兴睿[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,武汉430030

出　　处：《中华实验外科杂志》2011年第8期1306-1308,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：湖北省自然科学基金资助项目（2009CBB323）

摘　　要：目的构建DJ-1 shRNA真核表达载体，获得干扰质粒稳定转染的人肝癌HepG2细胞株。方法合成DJ-1基因干扰序列，定向插入真核表达载体pGenesil-1质粒并测序鉴定；干扰质粒经脂质体介导转染人肝癌HepG2细胞，G418（450mg／L）有限稀释法持续筛选8周后，扩增获得稳定转染的细胞株；荧光显微镜观察转染扩增效率；逆转录．聚合酶链反应（RT—PCR）和Westernblot检测稳定转染细胞株的DJ-1 mRNA和蛋白的表达。结果测序证明DJ-1干扰序列及读码框完全正确，G418筛选后扩增细胞中稳定转染扩增百分率高达75．00％。稳定转染细胞DJ-1 mRNA表达下调，DJ-1蛋白抑制率为98．78％（P〈0．05）。结论成功构建DJ-1 shRNA真核表达载体，DJ-1 shRNA质粒稳定转染HepG2细胞株的建立，为以DJ-1基因为靶点的人肝癌基因研究提供实验基础。Objective To construct the DJ-1 shRNA eukaryotic expression vectors and obtain the stably transfected HepG2 cell lines. Methods Targeting DJ-1 gene interference sequence was synthesized and inserted into pGenesil-1 vector. The recombinant RNA interference （RNAi） vectors were transfected into HepG2 cells with Lipofectamine 2000. The transfected cells were persistently screened under G418 （450 mg/L）, and isolated with a limited dilution for 8 weeks. Green fluorescence of the stably transfected HepG2 cell lines were observed under the fluorescence microscopy. The mRNA and protein expression of DJ-1 in the selected clones was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, respectively. Results Sequencing indicated that RNAi eukaryotic expression vectors targeting DJ-1 had correct reading frame and nucleotide sequence, and the positive rate of stable transfected HepG2 cells was more than 75% per clone. RT-PCR revealed that DJ-1 shRNA effectively silenced mRNA expression level of DJ-1. Western blotting discovered that DJ-1 shRNA obviously suppressed protein expression level of DJ-1 with the inhibition rate being 98.78% （P 〈 0. 05）. Conclusion DJ-1 shRNA eukaryotic expression vectors were successfully constructed, and the establishment of stably transfected HepG2 cell lines provided a original route for exploring the mechanism of DJ-1 in human hepatocellular carcinoma cells further.

关 键 词：DJ-1 RNA干扰 癌 肝细胞 

分 类 号：R735.7[医药卫生—肿瘤]