稳定高表达CYP2E1基因肝癌HepG2细胞系的构建  

Establishment of hepatocellular carcinoma cell line HepG2 stably over-expressing CYP2E1

作　　者：熊永福[1] 闫再华 杨召[1] 李敬东[2] 

机构地区：[1]川北医学院,四川南充637007 [2]川北医学院附属医院

出　　处：《山东医药》2015年第7期8-10,I0002,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81370531)

摘　　要：目的构建稳定高表达CYP2E1基因的肝癌HepG2细胞系。方法通过NCBI查询CYP2E1的编码序列,设计并构建表达质粒p LV(Exp)-Puro-CMVm-CYP2E1,用载体对应的慢病毒包装质粒(p MDLg/pRRE、pRSVREV和p MD2.G)共同转染293T细胞。利用携带CYP2E1基因的慢病毒(Lenti-CYP2E1-e GFP-Puro)感染HepG2细胞系,采用嘌呤霉素抗性筛选方法构建高表达CYP2E1肝癌HepG2细胞系。增强绿色荧光蛋白检测转染情况,荧光定量PCR及Western blotting检测HepG2、空载及转染CYP2E1基因的HepG2细胞系中CYP2E1 mRNA和蛋白。结果 HepG2细胞系均成功转染CYP2E1基因。HepG2、空载及转染CYP2E1基因的HepG2细胞系中CYP2E1mRNA相对表达量分别为1.02±0.06、1.06±0.05、7.42±0.07,蛋白相对表达量分别为0.26±0.02、0.29±0.01、1.61±0.08,转染CYP2E1基因的HepG2细胞系与前两者比较,P均<0.05。结论通过慢病毒转染成功构建了稳定高表达CYP2E1基因的肝癌HepG2细胞系。Objective To establish a hepatocellular carcinoma cell line HepG 2 which highly expressing CYP 2E1 stab-ly.Methods The coded sequence （CDS） of CYP2E1 was searched in NCBI website, and lentiviral vector （pLV（Exp）-Puro-CMVm-CYP2E1） was designed and constructed .293T cells were co-transfected with the vector corresponding lentivi-ral packaging plasmid （ pMDLg/pRRE, pRSV-REV and pMD2.G）.Lentiviral （ Lenti-Cyp2e1-eGFP-Puro） carrying CYP2E1 gene was used to trasfect HepG2 cell line, and puromycin-resistance screening were performed to establish the HepG2 cell line that highly expressed CYP 2E1.The transfection was detected through enhanced green fluorescent protein , q-PCR and Western blotting was used to detect the expression of CYP 2E1 mRNA and protein in HepG2, no-load HepG2 cell line and HepG2 cell line transfected by CYP2E1 gene.Results HepG2 cell line all transfected CYP2E1 gene suc-cessfully.The relative expression of CYP2E1 mRNA in HepG2, no-load HepG2 cell line and HepG2 cell line transfected by CYP2E1 gene was 1.02 ±0.06, 1.06 ±0.05 and 7.42 ±0.07, the protein expression of each group was 0.26 ±0.02, 0.29 ±0.01 and 1.61 ±0.08 respectively.HepG2 cell line transfected by CYP2E1 gene had significant differences as compared with the former two groups （ all P＆lt;0.05 ） .Conclusion The hepatocellular carcinoma cell line HepG 2 stably and highly expressing CYP2E1 gene is successfully constructed by lentiviral transfection .

关 键 词：CYP2E1基因 慢病毒载体 肝癌 HEPG2细胞 

分 类 号：R73[医药卫生—肿瘤]