摘要
为构建一种由人端粒酶逆转录酶启动子(hTERTp)调控的能够在肿瘤细胞中特异性增殖的新型肿瘤基因治疗溶瘤腺病毒载体系统,利用基因重组技术将hTERTp插入5型腺病毒E1A基因上游,构建肿瘤特异性增殖腺病毒AdSU,使其携带绿色荧光蛋白报告基因.同时构建增殖缺陷型腺病毒Ad-EGFP作为对照.通过Westernblot分析表明该特异性增殖腺病毒AdSU-EGFP在肿瘤细胞中特异性表达EIA.而通过荧光显微镜观察两者增殖实验发现,AdSU-EGFP在正常成纤维细胞株BJ及原代子宫成纤维细胞中无增殖,在肝癌细胞株MH-CC和肺癌细胞株A549中,则可见病毒增殖并裂解细胞的现象.由此可认为成功构建了肿瘤基因治疗的特异性溶瘤腺病毒载体系统AdSU,该系统能够在肿瘤细胞中特异性增殖并表达所携带的外源基因,这对肿瘤的定向基因治疗有着重要意义.
To develop a novel gene therapeutic oncolytic adenovirus in which the hTERT promoter was introduced and used to regulate adenoviral E1A gene. A novel cancer-specific replication-competent adenovirus named AdSU was constructed by employing the human telomerase reverse transcriptase （hTERT） promoter to drive the expression of adenovirus E1A gene and by cloning the EGFP reporter gene into the adenovirus genome. The non-replicative adenovirus named Ad-EGFP was constructed as the control at the same time. The selective replication of AdSU-EGFP in tumor cells was investigated by virus proliferative test and Western blot assay. By western blot assay, E1A protein was positive in cancer cells （ MHCC, A549） infected with AdSU-EGFP, but negative in normal ceils （ BJ, uterine fibroblast）. Under the fluorescent microscope, a few normal cells expressed EGFP and emitted fluorescence, and no phagocytic plagues appeared from 3 - 10 days after infected with AdSU-EGFP. However, when a few cancer cells were infected with AdSU - EGFP and emitted fluorescence after 3 days, the fluorescence emission ceils spread within a large area after 7 days. So we can make a conclusion that a novel cancer-selective gene therapeutic oncolytic adenovirus vector system named AdSU, which can selective replicate and express therapeutic genes in cancer cells, was constructed. It is a promising system for targeted cancer gene therapy.
出处
《南京师大学报（自然科学版）》
CAS
CSCD
北大核心
2008年第2期92-96,共5页
Journal of Nanjing Normal University(Natural Science Edition)
关键词
基因治疗
溶瘤腺病毒
肿瘤
端粒酶启动子
gene therapy, oncolytic adenovirus, tumor, telomerase promoter
