MDA-7基因的腺病毒载体构建及对肿瘤细胞凋亡的影响  

Construction of tumor-specific proliferation-deficient adenovirus vector and its primary study on selective expression in tumor cells

作　　者：王艳[1] 浦颖艳[2] 方琳[2] 孙丽君[2] 胡晓翠[2] 邱红[3] 苏长青[2] 曹祥荣[2] 江淑芳[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013 [2]南京师范大学生命科学学院,江苏南京210046 [3]中国人民解放军第八一医院检验科,江苏南京210002

出　　处：《江苏大学学报（医学版）》2008年第3期205-208,共4页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:构建一种端粒酶启动子调控目的基因MDA-7/hIL24表达的复制缺陷型腺病毒,并初步探索其抗肿瘤活性。方法:通过基因操作技术将启动子(hTERTp)+目的基因(MDA-7)插入到5型腺病毒E1A区域,构建复制缺陷型腺病毒AdTP-mda7;同时构建对照病毒AdTP-EGFP。TCID50法测定病毒滴度。应用蛋白质印迹法检测MDA-7在肿瘤细胞株中的表达状态;通过结晶紫染色实验检测两种病毒对肿瘤细胞的杀伤差异。结果:成功构建携带目的基因的腺病毒载体,PCR鉴定正确;蛋白质印迹结果表明MDA-7选择性地在多种肿瘤细胞株中表达,诱导肿瘤细胞凋亡,如A549,SGC-7901等,在以相同MOI值感染原代成纤维细胞时,原代细胞中没有检测到MDA-7表达;结晶紫染色试验结果表明AdTP-mda7对肿瘤细胞株的杀伤能力明显高于对照病毒AdTP-EGFP。结论:成功构建复制缺陷型腺病毒AdTP-mda7,并证实MDA-7蛋白在肿瘤细胞中过表达诱导多种肿瘤细胞株发生凋亡。Objective： To develop a proliferation-deficient adenovirus for gene therapy in which the hTERT promoter was introduced to regulate the expression of the target gene in tumor cells. Methods： A tumor-specific proliferation-difficient adenovirus vector was constructed by employing the human telomerase reverse transcriptase （hTERT）promoter to drive the expression of adenovirus-mediated target gene; at the same time AdSuTP-EGFP was constructed as control. Titer was obtained by TCID50 method. The expression of MDA-7 was detected by western blot and the appearance of senescence was assessed by violet staining. Cells were photographed 2h after staining. Results： Two adenovirus vectors including target gene were successfully constructed and identified by PCR technology. Western blot analysed that MDA-7 selectively expressed in tumor cell lines MHCC, SGC7901, A549, and so on, and induced apoptosis,whiie primary lung fibroblast was transduced at equal MOI level and did not express detectable levels of MDA-7 ; violet staining test verified directly that killing effect of AdTP-mda7 was great stronger than AdTP-EGFP, supporting that one of the functions of MDA-7 was apoptosis induction. Conclusion： proliferation-deficient adenovirus, AdTP-mda7, was successfully constructed, simultaneously verified that the overexpression of MDA-7 in tumor cells induced tumor cell apoptosis, which was a novel gene-virus therapeutic system and significant for targeted tumor gene therapy.

关 键 词：复制缺陷型腺病毒 端粒酶启动子 肿瘤基因治疗 

分 类 号：R392.12[医药卫生—免疫学]