选择性杀伤CEA阳性结直肠癌细胞的条件复制型腺病毒载体的构建及应用  

Construction and application of conditionally replicative adenovirus for selective cytotoxicity in CEA positive colorectal cancer cells

作　　者：王梦筠[1] 韦芳[1] 王慧萍[1] 季迅达[1] 陈霞芳[1] 李惠明[1] 冯烨[1] 王煜非[1] 黄倩[1] 

机构地区：[1]上海交通大学第一人民医院中心实验室,上海200080

出　　处：《上海交通大学学报（医学版）》2009年第7期802-807,共6页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家重点基础研究发展计划("973"计划) (2004CB518804);国家自然科学基金(30325043; 30428015)~~

摘　　要：目的构建能选择性杀伤表达癌胚抗原(CEA)的结直肠癌细胞的条件复制型腺病毒载体。方法PCR扩增CEA基因5’端调控序列,构建以EGFPLuc作为报告基因的表达质粒pCEA-EGFPLuc。利用脂质体将pCEA-EGFPLuc导入CEA阳性和阴性细胞中,通过检测增强型绿色荧光蛋白(EGFP)表达和荧光素酶(luciferase)活性,评价CEA启动子活性。构建由CEA启动子调控的E1A,并携带HSVtk基因的条件复制型腺病毒Ad.CEA-E1A/CMV-TK,分别感染CEA阳性肿瘤细胞(Lovo和SW620)和阴性肿瘤细胞(HeLa),通过E1A表达和细胞存活率检测,评价该病毒对CEA阳性肿瘤细胞的选择性杀伤效果及联合应用环氧鸟苷(GCV)后的协同效应。结果CEA启动子具有较好的选择性和较高的活性。Ad.CEA-E1A/CMV-TK只在CEA阳性的肿瘤细胞中表达E1A。感染Ad.CEA-E1A/CMV-TK后,Lovo和SW620细胞的存活率分别为(36.72±2.49)%和(39.82±4.76)%,均显著低于HeLa细胞的(87.44±2.76)%(P<0.01);联合GCV对Lovo和SW620的杀伤效果增加,细胞存活率分别为(17.26±3.65)%和(23.93±5.40)%,显著低于未加GCV的(36.72±2.49)%和(39.82±4.76)%(P<0.01)。结论由CEA调控复制的Ad.CEA-E1A/CMV-TK对CEA阳性的结直肠癌细胞具有选择性杀伤作用,联合GCV后杀伤效果明显增强。Objective To construct a new conditionally replicative adenovirus （CRAds） targeting carcinoembryonic antigen （CEA） positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive（ Lovo and SW620）and negative tumor cells（HeLa） were infected with Ad. CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad. CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were （36.72 ± 2.49）% and （39.82±4.76）%, respectively, significantly lower than that of Hela[（87. 44 ± 2. 76）%] （P 〈 0.01）. When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of （ 17.26± 3.65） % and （23.93± 5.40） %, respectively, significantly lower than those without GCV[ （36.72 ± 2.49） % and （39.82 ±4.76） %, respectively] （ P 〈 0.01）. Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer ceils, and the effect can be enhanced when combined with GCV

关 键 词：结直肠癌 癌胚抗原 启动子 重组腺病毒 E1A基因 HSVtk自杀基因 

分 类 号：R735.3[医药卫生—肿瘤] Q782[医药卫生—临床医学]