携带反义甲胎蛋白增强子/单纯疱疹病毒胸苷激酶基因的产病毒细胞系的建立及对肝癌细胞的靶向杀伤效应  

Construction of virus producer cell lines bearing antisense AFP enhancer/HSV-TK gene and its specific effect on hepatoma cells

作　　者：茅幼霞 陈迟[2] 刘景丰[3] 

机构地区：[1]上海职工医学院病理教研室,200237 [2]上海第二医科大学附属仁济医院消化疾病研究所 [3]福建医科大学附属第一医院外科

出　　处：《中华实验外科杂志》2005年第8期944-946,共3页Chinese Journal of Experimental Surgery

摘　　要：目的建立对肝癌细胞具有靶向调控的高感染性和高分泌性产病毒细胞系。方法将重组反义甲胎蛋白增强子/单纯疱疹病毒胸苷激酶基因逆转录病毒载体(pL/TK/AFE/SN)转染至PA317包装细胞,经G418(400mg/L)筛选、病毒滴度测定。以病毒上清感染培养的Hep3B肝癌细胞和Hela宫颈癌细胞,并抽提细胞基因组DNA作Southernblot分析;观察GCV(100mg/L)对细胞生长的影响。结果转染pL/TK/AFE/SN的PA317细胞经G418筛选2周获阳性克隆;病毒滴度为5.4×104～3.8×105CFU/ml。Southern杂交证实前病毒已完整整合于PA317细胞,并稳定整合于Hep3B肝癌细胞和Hela宫颈癌细胞中。导入AFE/TK基因的Hep3B肝癌细胞经GCV作用后的存活率在2、4、6d时分别降低35%、87%、95%,生长明显受抑制(F=7.23,P<0.01)。结论携带AFE/TK基因的产病毒细胞系能够成功地将AFE/TK基因转移到感染细胞中。Objective To construct, virus producer cell lines that could be target-controlled in the hepatoma ceils and to lay down the experimental basis for further research. Methods Recombinant retrovirus vector bearing antisense AFP enhancer/TK gene （pL/TK/AFE/SN） wastransfected into PA317 packaging cells. The virus-infected PA317 ceils were selected in medium with 400 mg/L G418. The virus titer of PA317 cells was analyzed on NIH3T3 ceils. Highest titer PA317 cells clones containing provirus were selected and their viruses were used to infect Hep3B hepatoma cell line and Hela cervical carcinoma cell line. The constructs containing provirus in infected cells were detected by Southern blot and their effects on biological behavior of infected cells were observed by HSV-TK/GCV system. Results The virus titer mainly ranged from 5.4 × 10^4 CFU/ml-3.8 × 10^5 CFU/ml. Southern-blot analysis revealed that the infected PA317 cells, Hep3B hepatoma cells and Hela cervical carcinoma cell line contained correct provirus. In the presence of GCV, the growth of Hep3B hepatoma cells infected with antisense AFE/TK fusion gene was significantly suppressed as compared with others （F = 7.23, P〈 0.01 ）. Conclusion The virus producer cell lines bearing AFE/TK gene established in our experiments could faithfully transfer the AFE/TK gene to the infected cells.

关 键 词：反义甲胎蛋白增强子 单纯疱疹病毒 胸苷激酶 产病毒细胞系 肝癌 肿瘤细胞 靶向杀伤效应 

分 类 号：R735.7[医药卫生—肿瘤]