大鼠肝癌细胞HSV-tk/GCV自杀基因系统的构建及其旁观者效应  

Construction of HSV-tk/GCV suicide gene system for hepatocarcinoma cells and its bystander effect in rats

作　　者：谭宇蕙[1] 吴映雅[1] 杜标炎[2] 胡岳山[1] 赵鹏[2] 李杰芬[3] 

机构地区：[1]广州中医药大学生化教研室,广东省广州市510405 [2]广州中医药大学病理教研室,广东省广州市510405 [3]广州中医药大学中医基础实验中心,广东省广州市510405

出　　处：《世界华人消化杂志》2005年第17期2069-2073,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30171201;广州市科技计划项目;No.2002J1-C7041~~

摘　　要：目的:构建针对大鼠肝癌细胞的HSV-tk/GCV(以下简称tk/GCV系统)自杀基因治疗系统,建立比较tk/GCV系统作用效果及其旁观者效应大小的细胞模型和检测方法.方法:用包装细胞PT67/tk分泌的重组逆转录病毒的活性颗粒感染大鼠肝癌CBRH7919细胞,用G418筛选3wk获得高耐药性的重组子CBRH7919/tk,扩大培养.提取CBRH7919(tk-),CBRH7919/tk(tk+)细胞的DNA,把HSV-tk基因片段用Dig标记为探针后,与2种DNA及阳性对照DNA进行点杂交,以检测tk+细胞的DNA是否整合有HSV-tk基因.用0.1、1、10、50、100mg/L的GCV体外作用于tk-、tk+细胞,MTT检测存活率.用GCV体外分别作用于含不同比例tk+细胞的tk+、tk-混合细胞,MTT检测存活率,比较分析各组旁观者效应大小,确定后续实验的合适tk+比例.结果:用G418筛选3wk后获到高耐药性的CBRH7919/tk(tk+)重组细胞.HSV-tk基因片段用Dig标记为探针后进行点杂交,检测到tk+细胞DNA上已整合有HSV-tk基因,而tk-细胞DNA不含HSV-tk基因.GCV对tk+细胞生长抑制和细胞毒作用明显,而且有浓度、时间依赖性,而tk-细胞对GCV不敏感,说明HSV-tk基因已表达且表达产物具有生物学活性.GCV对含5%和10%tk+的混合细胞杀伤率分别为6.0%和25.2%,结果表明5-10%的tk+混合比例下自杀基因系统自身的旁观者效应较低,而且用MTT检测旁观者效应大小时灵敏度比较高,可作为后续研究中药活性成分对旁观者效应增效作用的tk+、tk-混合比例.结论:成功构建了针对大鼠肝癌的HSV1-tk/GCV自杀基因治疗系统,建立了体外快速筛选旁观者效应增效药物的稳定有效的细胞模型和检测方法.AIM： To construct the therapeutic system of HSV-tkl GCV suicide gene for hepatocarcinoma cell line, and to establish a cell model and method to examine the effect of HSV-tklGCV system and its bystander effect. METHODS： CBRH7919 cells was infected by the active retrovirus particle excreted by the packaging cell line PT67/tk, and then a high anti-G418 clone named CBRH7919/tk was obtained by G418 screening for 3 wk. The total DNAs of CBRH7919 and CBRH7919/tk cells were extracted. The cDNA fragments of HSV-tk were labeled with Digoxin and then hybridized with the DNAs of CBRH7919, CBRH7919/tk cells and positive controls. The CBRH7919 and CBRH7919/tk cells were treated with different concentrations （0.1, 1, 10, 50, and 100 mg/L） of GCV, respectively. The mixture with different ratios of tk positive and negative cells was also treated with GCV. The survival rates of the cells were examined by MTT assay. The bystander effect was analyzed to determine a suitable proportion of tk positive cells for the successive experiments. RESULTS： A high anti-G418 clone named CBRH7919/ tk （tk^＋） was obtained. The HSV-tk gene was detected in the CBRH7919/tk cells, while it was not detected in the CBRH7919 cells. GCV showed significant cytoxicity and inhibition on the proliferation of CBRH7919/tk cells in a concentration- and time-dependent manner. The CBRH7919（tk） cells manifested low sensitivity to GCV. The killing rate of GCV to the mixtures containing 5% and 10% tk positive cells were 6.0% and 25.2%, respectively. The sensitivity of MTT assay in the detection of the bystander effect was relatively high, and the bystander effect was low when the mixture contained 5-10% tk positive cells. CONCLUSION： The therapeutic system of HSV tk/GCV suicide gene for hepatocarcinoma cells in rats is successfully constructed, and the mixed cells containing 5-10% tk positive ones can be used for further study.

关 键 词：HSV1-tk/GCV自杀基因系统 肝癌 大鼠 旁观者 效应 

分 类 号：R735.7[医药卫生—肿瘤]