【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。更多还原

【Abstract】 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,while 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.更多还原