Pan Yu-qin, He Bang-shun, Zhu Chan, Qu Li-li, Xu Xiong-fei, Wang Shu-kui
Nanjing Medical University, Nanjing, China.
Zhonghua Zhong Liu Za Zhi. 2011 Nov;33(11):816-21.
To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.
The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.
The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.
The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.
探讨IGF2印记系统在肿瘤靶向基因治疗中的可行性。
从小鼠基因组DNA中通过PCR扩增小鼠H19增强子、DMD和启动子H19，然后克隆到质粒pDC312中。通过PCR扩增EGFP和DT-A片段并克隆到重组质粒中，随后将穿梭质粒与腺病毒载体Ad5一起转染到HEK293细胞中，即Ad-EGFP和Ad-DT-A。应用腺病毒六邻体基因表达来确认腺病毒感染的存在。通过荧光显微镜检测IGF2印记系统的效果。将重组腺病毒载体转染到癌细胞后，用RT-PCR和Western印迹分别显示IGF2印记缺失（LOI）和IGF2印记维持（MOI）。对HCT-8（LOI）进行MTT和流式细胞术评估其抗肿瘤效果。体外将Ad-DT-A转染人乳腺癌细胞系MCF-7（MOI）和人正常胃上皮GES-1（MOI）细胞系。通过将Ad-DT-A注射到携带HCT-8肿瘤的裸鼠中检测其抗肿瘤效果。
仅在HCT-8肿瘤细胞系中可见EGFP蛋白、DT-A mRNA和DT-A蛋白表达呈阳性。Ad-DT-A感染导致HCT-8细胞生长明显受到抑制，与对照组相比为（75.4±6.4）%，并增加了HCT-8细胞的凋亡百分比（20.8±5.9）%。将重组腺病毒注射到携带HCT-8肿瘤的裸鼠中进一步证实了其抗肿瘤效果，结果显示Ad-DT-A抑制肿瘤生长，抑制率为36.4%。
成功构建了携带IGF2印记系统和DT-A基因的重组腺病毒，而Ad-DT-A可有效杀死显示IGF2印记缺失的肿瘤细胞。它可能在未来基于IGF2印记缺失的恶性肿瘤靶向基因治疗中发挥重要作用。
