重组表达质粒pcDNA3/HIF-1α的构建及其在人肝癌细胞株HepG2的稳定表达  

Construction of pcDNA3/HIF-1α Expression Plasmid and its Stable Expression in Liver Cancer Cell Line HepG2

作　　者：曾少波[1] 赵艳梅[1] 江斌[1] 菅志远[1] 严斌[1] 刘炯[1] 

机构地区：[1]郧阳医学院附属太和医院普通外科,湖北十堰442000

出　　处：《郧阳医学院学报》2009年第2期125-127,F0002,共4页Journal of Yunyang Medical College

摘　　要：目的:构建缺氧诱导因子(HIF-1α)基因的表达质粒,经脂质体转染人肝癌细胞株HepG2,建立稳定表达HIF-1α基因的细胞模型,为肝癌研究提供有效工具。方法:采用RT-PCR方法扩增肝癌细胞株HepG2内HIF-1α基因功能区片段,克隆至真核表达载体pcDNA3上,转染HepG2细胞后,经G418筛选,建立稳定表达HIF-1α基因的细胞株,免疫荧光染色和Western-blot分析HIF-1α蛋白的表达。结果:通过酶切鉴定,凝胶电泳分析,两条带大小分别位于5.3kb和2.55kb,与预期结果符合,序列测定证实HIF-1α与GeneBank公布的序列一致。经脂质体包裹转染HepG2后,免疫荧光染色和Western-blot分析证实HIF-1α蛋白的表达明显上调。结论:经脂质体途径成功构建稳定表达HIF-1α的细胞模型,为肝癌研究提供了一个有效的工具。Objective To construct the plasmid of HIF-1α and transfect it into human liver cancer cell line HepG2 with liposome ,to establish the cell model stably expressing HIF-1α and provide an effective tool for liver cancer research. Methods The function region of HIF-1α mRNA in HepG2 was amplified with RT-PCR and subeloned into the vector peDNA3 ,the cell lines that stably expressed HIF-1α were selected with G418 peDNA3/HIF-1α after transfeeted into HepG2, the expression of HIF-1α protein was detected with Western blot and immunofluorescenee staining. Results The constructed expression plasmid was identified with restriction enzyme digestion and gel elecrrophoresis, two DNA bands at 5.3kb and 2.55kb were found, respectively, which was in accordance with expected results, the sequence of cloned HIF-1α gene was the same as that reported in GeneBank. Eukaryotie expression plasmid was transfected into cultured HepG2 cells with liposome, the expression of HIF-1α protein detected with Western blot and immunofluorescence staining was significantly increased. Conclusion HepG2 with stable HIF-1α expression was successfully established,which may be an effective tool for liver cancer research.

关 键 词：HEPG2 缺氧诱导因子 基因转染 

分 类 号：Q78[生物学—分子生物学]