反义AFP增强子/TK基因重组逆转录病毒载体的构建及在肝癌细胞中的专一性表达  

Construction of recombinant retrovirus vector bearing antisense AFP enhancer/TK gene and its hepatocyte-specific expression

作　　者：茅幼霞 陈迟[2] 徐卫国[3] 

机构地区：[1]上海职工医学院病理教研室,上海200237 [2]上海第二医科大学附属仁济医院消化疾病研究所,上海200237 [3]华中科技大学同济医学院同济医院外科,湖北武汉430030

出　　处：《中国现代医学杂志》2005年第6期851-854,860,共5页China Journal of Modern Medicine

摘　　要：目的构建在肝癌细胞中特异性高表达的重组逆转录病毒载体,观察其对体外培养肝癌细胞生物学行为的影响。方法应用DNA重组技术将α-甲胎蛋白増强子(AFE)核心区DNA与单纯性疱疹病毒胸苷激酶(TK)基因融合,反向插入逆转录病毒载体pLXSN,重组体命名为pL/TK/AFE/SN;经PA317细胞包装、G418筛选、病毒滴度测定,挑选滴度高的抗性克隆细胞株扩增培养,收获病毒上清,感染体外培养的Hap3B肝癌细胞和Hela宫颈癌细胞。用Southernblot、Northernblot检测转染细胞的DNA、mRNA,以羟甲基无环鸟苷(GCV)对细胞的杀伤毒性检测外源基因在转染细胞中的表达。结果酶切及Southernblot证实插入pL/TK/AFE/SN的反义AFE/TK基因大小、方向及克隆位点正确。达到一定的产病毒滴度。Northernblot分析显示Hep3B肝癌细胞有TK基因mRNA水平表达;应用GCV后,导入反义AFE/TK基因的Hep3B肝癌细胞生长明显受抑制(P<0.01)。结论成功构建了含反义AFE/TK基因的重组逆转录病毒载体并包装出具有感染能力的假型逆转录病毒;重组载体所含的反义AFP増强子能调控外源性TK基因特异地在Hep3B肝癌细胞中高表达。To construct antisense recombinant Retrovirus vector that would express in hepatoma cells efficiently and specifically and to observe its affection on biological behavior of hepatoma cells. A fusion gene of alpha- fetoprotein enhancer(AFE) core region fragment and Herpes simplex virus ThymidineKinase(TK) gene, which was produced using recombinant DNA technique was cloned inversely into retrovirus vector pLXSN, resulting in a recombinant vector pL/TK/AFE/SN. pL/TK/ AFE /SN and pLXSN were infected into PA317 packaging cells with calcium phosphate method. The virus-infected PA317 cells were selected in medium with 400 μg/mL G418 and infected into Hep3B hepatoma cell line and Hela cervical carcinoma cell line were infected in medium containing pseudotype retrovirus. The expression level of TK gene in cell infected with antisense AFE /TK fusion genewere detected by Northern blot. Growth rate of infected cell were observed by ganciclovir(GCV). The recombinant retrovirus vector harbouring AFE /TK fusion gene was confirmed correctly by restriction endonuclease digestion and southern hybridization. The recombinant retroviral vector pL/TK/ AFE /SN after packaging PA317 cell and expressed in NIH3T3 cell as showed, have higher infectivity. The mRNA of TK gene was expressed in Hep3B hepatoma cells as showed by Northern blot analyze. In the presence of GCV,Growth rate of Hep3B hepatoma cells infected with antisense AFE /TK fusion gene were significantly suppressed compared with others(P <0.01). [Conclusion] The recombinant vector bearing antisense AFE /TK gene was successfully constructed, the packaged pseudotype retrovirus can infect mammalian cells, and the mRNA of TK gene is specifically expressed in Hap3B hepatoma cells. This research may lay a basis for targeted therapy of hepatoma.

关 键 词：AFP增强子 逆转录病毒载体 肝癌细胞 

分 类 号：R735.7[医药卫生—肿瘤]