IGF2基因组印记介导结直肠癌靶向治疗的实验研究  

Targeted therapy for colorectal cancer based on the loss of insulin-like growth factor 2 imprinting

作　　者：聂珍琳[1,2] 潘玉琴[2] 何帮顺[2] 许晔琼[2] 李瑞[2] 高天翼[2] 王书奎[1,2] 

机构地区：[1]南京医科大学附属南京医院/南京市第一医院检验科,南京210006 [2]南京医科大学附属南京医院/南京市第一医院中心实验室,南京210006

出　　处：《临床检验杂志》2014年第1期9-14,共6页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金面上项目(81172141)

摘　　要：目的探讨携带胰岛素样生长因子2印记系统(IGF2 imprinting)的重组腺病毒靶向治疗结直肠癌的可行性。方法用PCR扩增H19、enhancer、CTCF、EGFP和E1A片段并克隆至pDC-315中构建成重组腺病毒穿梭质粒,分别与腺病毒骨架共转染包装成腺病毒Ad315-EGFP和Ad315-E1A。Ad315-EGFP分别转染GES-1、HCT-116、HCT-8和HT-29细胞,荧光显微镜观察各组细胞中EGFP的表达。以H101作为阳性对照,用RT-PCR及western blot检测Ad315-E1A和H101转染后各组细胞中E1A基因和蛋白质的表达;用MTT法和流式细胞术检测Ad315-E1A体外抗肿瘤效应。构建皮下移植瘤裸鼠模型,检测Ad315-E1A的体内抑瘤作用。结果 Ad315-EGFP转染各组细胞,EGFP仅在IGF2 LOI细胞HCT-8和HT-29中表达;各组细胞经Ad315-E1A转染后,E1A基因和蛋白质仅在LOI细胞(HCT-8和HT-29)中表达;HCT-8细胞经Ad315-E1A 10 PFU/cell转染72 h后,增殖活力降低至(53.4±6.4)%,凋亡率升高至(23.9±3.7)%;经H101转染的各组细胞,E1A基因和蛋白质仅在p53突变的细胞HT-29中表达;多点注射Ad315-E1A治疗HCT-8移植瘤显示其抑瘤率达32.1%。结论成功构建了携带IGF2印记系统的重组腺病毒;重组腺病毒Ad315-E1A能够有效杀伤IGF2 LOI结直肠癌细胞。Objective：To investigate the feasibility of recombinant adenovirus carried the insulin-like growth factor 2 （IGF2） imprinting system in the targeted therapy of colorectal cancer. Methods：The mouse H19 enhancer, CTCF, EGFP and  E1A fragments were amplified by PCR and cloned into the plasmid pDC-315 to construct the recombinant shuttle plasmids. Then the plasmids and the adenovirus vectors were transfected into HEK293 cells to construct adenovirus Ad315-EGFP and Ad315-E1A. Ad315-EGFP and Ad315-E1A were transfected respectively into HCT-8 （LOI）, HT-29 （LOI）, HCT-116 （MOI） and GES-1 （MOI） cells. The cells transfected Ad315 EGFP were detected the EGFP expression by fluorescence microscope. The cells transfected Ad315 E1A were detected the expressions of E1A gene and protein by RT-PCR and western blot, respectively. The in vitro anti-tumor effect of Ad315-E1A was assessed by MTT and flow cytometry, respectively. Meanwhile, the in vivo anti-tumor effect was evaluated by injecting the Ad315-E1A into the nude mice carrying HCT-8 tumors. Results：Among the cells transfected Ad315 EGFP, only IGF2 loss of imprinting （LOI） cells, such as HCT-8 and HT-29, expressed EGFP. Similarly, among the cells transfected Ad315-E1A, only HCT-8 and HT-29 cells expressed E1A mRNA and protein. After HCT-8 cells were transfected 10 PFU/cell of Ad315-E1A for 72 hours, their proliferation rate reduced to （53.4±6.4）%, and the apoptosis rate increased to （23.9±3.7）%. After the Ad315-E1A was injected into the nude mice carrying HCT-8 tumors, the inhibition rate of tumor formation increased to 32.1%. In addition, among the cells transfected H101 （positive control）, only HT-29 cells expressed E1A mRNA and protein. Conclusion：The recombinant adenovirus carried the IGF2 imprinting system was successfully constructed, and the Ad315-E1A could effectively kill the colorectal cancer cells with IGF2 LOI.

关 键 词：基因印记 重组腺病毒 胰岛素样生长因子2 结直肠癌 靶向治疗 

分 类 号：R735.3[医药卫生—肿瘤]