中文摘要：

目的：构建携带IGF2印迹系统的腺病毒载体,并验证其在肿瘤细胞及正常细胞中的功效,为IGF2印迹在肿瘤靶向治疗中的应用提供理论基础。方法：将人源的IGF2印迹系统启动子H19、增强子enhancer及甲基化区域CTCF克隆至穿梭质粒pDC-312中构建IGF2基因印迹系统,从pDC-315-EGFP质粒中扩增出EGFP片段插入到构建好的IGF2印迹系统中,然后与腺病毒骨架Ad5通过脂质体Lipofectamine 2000介导共转染HEK293细胞,包装成有感染能力的腺病毒Ad-H19-CTCF-enhancer-EGFP,命名为Ad-EGFP;构建好的腺病毒分别感染IGF2基因印记保持的细胞MCF-7和GES-1及IGF2基因印迹丢失的细胞HRT-18,荧光显微镜下观察EGFP在三种细胞中表达的差异。结果：转染腺病毒载体的HEK293细胞表达EGFP随着时间逐渐增强,并且出现明显的细胞病变效应,EGFP在HRT-18细胞中有大量表达,在MCF-7和GES-1细胞中不表达或仅有少量表达。结论：成功构建了携带IGF2基因印迹系统的腺病毒载体,证明其在IGF2基因印迹丢失的肿瘤细胞中特异性的表达,在正常细胞及IGF2基因印迹保持细胞中不表达,为IGF2基因印迹系统应用于肿瘤细胞的靶向治疗提供了理论基础。

英文摘要：

Objective：To construct recombinant adenovirus carrying IGF2 imprinting system and to investigate the expression of the reporter gene EGFP of the recombinant adenovirus in tumor cells and normal cells. Methods： A IGF2 imprinting system was constructed via inserting promoter H19,CCCTC binding factor （CTCF）, enhancer of IGF2 and reporter gene EGFP into shuttle plasmid pDC-312. Then the recombinant plasmid and Ad5 skeleton were co-transfected into HEK293 cells for packaging. Recombinant adenovirus Ad-H19-CTCF-enhancer-EGFP （short for Ad-EGFP） was detected by PCR and the infection titer was monitored. The expression of the reporter gene EGFP of Ad-EGFP was tested after infecting IGF2 loss of impriting （LOI）cancer cell line HRT-18 and IGF2 maintenance of imprinting （MOI）cancer cell line MCF-7and normal cell line GES-1. Results： The IGF2 imprinting system was cloned into the adenovirus successfully. Viral titer was about 5.5×l09pfu/ml. Fluorescence microscope observation demonstrated that the IGF2 impriting system works in IGF2 LOI cancer cells but not in IGF2 MOI cells. Conclusions：The constructed recombinant adenovirus containing IGF2 impriting system provides a foundation for investigating the application of the system in tumor targeted therapy.