文档介绍：MDA7基因的腺病毒载体构建及对肿瘤细胞凋亡的影响
(作者:___________单位: ___________邮编: ___________)
【摘要】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。
【关键词】复制缺陷型腺病毒端粒酶启动子肿瘤基因治疗
[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 : 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 was obtained by TCID50 expression of MDA7 was detected by western blot and the appearance of senescence was assessed by violet were photographed 2h after staining. Results: Two adenovirus vectors including target gene were essfully constructed and identified by PCR blot analysed that MDA7 selectively expressed in tumor cell lines , 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 essfully 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 target