FasL逆转录病毒转移体系的建立及其在肝癌细胞中的表达  

Development of retroviral vectors containing rat Fas ligand gene and FasL expression mediated by the vectors in hepatocellular carcinoma cells

作　　者：孙倍成[1] 王学浩[1] 钱建民[1] 张峰[1] 李相成[1] 许志祥[2] 张学光[2] 

机构地区：[1]南京医科大学第一附属医院肝胆外科,210029 [2]苏州医学院生物技术研究所

出　　处：《中华实验外科杂志》2001年第2期114-116,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省自然科学基金资助项目 (BK991 4 9);江苏省政府重点课题资助项目 (BJ980 2 5)

摘　　要：目的　研究Fas/FasL系统的生物学功能 ,探讨转FasL基因治疗肿瘤的可行性。方法将大鼠FasL全长cDNA亚克隆到逆转录病毒载体 pLXSN中 ,获得FasL正向单拷贝插入子pLXSN/FasL+ ,经PA3 17包装细胞包装 ,获得G418抗性克隆 ,命名为PA3 17/pLXSN FasL+ 细胞。结果　聚合酶链反应扩增证实PA3 17/pLXSN FasL+ 细胞有外源性FasL基因整合 ;检测病毒滴度为 4.7× 10 7/L ;病毒上清转染肝癌细胞株HepG 2、SMMC 772 1、CBRH 7919和RH 3 5后 ,流式细胞仪检测肝癌细胞表面FasL分子表达的阳性率分别为 97.3 %、99.1%、99.4%和 72 .3 % ,并证实Fas高表达细胞株HepG 2及CBRH 7919对FasL诱导的凋亡十分敏感。结论　将FasL基因导入逆转录病毒载体是一种行之有效的基因转移途径 ,可用来诱导Fas高表达细胞凋亡 。Objectives To study the biological function of Fas and Fas ligand system and the feasibility of treating tumor with transfecting FasL gene. Methods The rat Fas ligand complementary DNA was subcloned to the retroviral vector pLXSN to obtain pLXSN/FasL + recombinant with direct inserting then packaged with PA317 amphotropic packaging cells, anti G418 clones were acquired and named as PA317/pLXSN FasL + cells. Results There were FasL gene integration in PA317/ pLXSN FasL + cells detected by polymerase chain reaction. The titer of virus was 4.7×10 7/L. When the supernatant of the PA317/pLXSNFasL + cells was used to transfect hepatocellular carcinoma cells HepG 2, SMMC 7721, CBRH 7919 and RH 35, the FasL positive rate on the hepatocellular carcinoma cells was 97.3%, 99.1%, 99.4% and 72.3% respectively by FCM. The apoptosis in HepG 2 and CBRH 7919 cells with high levels of Fas expression was found. Conclusion Introduction of FasL gene to retroviral vectors was an effective way, which can be used to induce apoptosis in the cells with high levels of Fas expression and provide theoretical basis for the gene therapy of tumor.

关 键 词：FAS配体 基因表达 逆转录病毒科 肝肿瘤 脱噬作用 

分 类 号：R735.7[医药卫生—肿瘤]