基于IGF2基因组印迹丢失的5型重组腺病毒靶向治疗结直肠癌的实验研究

发布时间：2018-07-28 09:39

【摘要】：研究目的：构建携带胰岛素样生长因子2印迹(IGF2 Imprinting)系统的重组腺病毒,探讨其用于靶向治疗人结直肠癌(colorectal cancer, CRC)细胞株的可行性。研究方法：(1)使用PCR扩增人源IGF2 Imprinting相关的结构元件,包括H19启动子序列、enhancer及CTCF片段,并将其克隆到pDC-312载体中,最终构建IGF2Imprinting系统；(2)从质粒TopK和pEGFP-C1 vector中分别扩增出E1A和EGFP片段,并将其插入到含有IGF2 Imprinting系统的重组质粒中,用于构建重组腺病毒穿梭质粒；(3)Topl0感受态细胞通过扩增和纯化,将腺病毒穿梭质粒pDC312-E1A,骨架质粒Ad5通过脂质体Lipo2000共转染到人胚肾细胞(HEK293)中,包装成具有感染能力的腺病毒Ad-H19-CTCF-enhancer-E1A和Ad-H19-CTCF-enhancer-EGFP,分别命名为Ad312-E1A和Ad312-EGFP;(4)利用腺病毒Ad312-EGFP分别感染IGF2印记丢失(IGF2 LOI)的结直肠癌细胞HT-29和HCT-8及IGF2印迹正常(IGF2 MOI)的结直肠癌细胞SW480和正常人胃粘膜上皮细胞株GES-1,通过荧光显微镜观察不同细胞中EGFP的表达情况；(5)使用已上市的溶瘤腺病毒H101作为阳性对照,将Ad312-E1A和H101感染不同的细胞株,48 h后采用RT-PCR和Western Blot分别从转录和翻译水平检测各组细胞中E1A的nRNA及其蛋白的表达量；同时,采用CCK-8法和流式细胞仪用以检测腺病毒Ad312-E1A的体外杀伤肿瘤效应；(6)构建裸鼠腋下移植瘤模型,验证Ad312-E1A的体内杀伤肿瘤效应。研究结果：(1)成功转染了穿梭质粒pDC312-E1A和骨架质粒Ad5到HEK293细胞中,EGFP在48 h表达最强；10-13 d,细胞病变效应为阳性结果,通过3轮病毒的扩增达到了合适的病毒滴度(1×109 pfu/ml);(2)利用重组腺病毒Ad312-EGFP分别感染不同的细胞株,48 h观察,IGF2 LOI的细胞株(HT-29, HCT-8)出现了明显的荧光,即表达了EGFP;(3)利用重组腺病毒Ad312-E1A分别感染不同的细胞株,48 h后发现E1A的mRNA和蛋白仅仅在IGF2 LOI细胞(HT-29、HCT-8)中表达；与正常对照PBS组相比,72 h后10 pfu/cell感染的IGF2 LOI细胞(HT-29、HCT-8)增殖活力分别下降(69.7±5.7)%和(65.2±7.1)%,凋亡率达到(33.4±4.1)%和(29.5±2.7)%；溶瘤腺病毒H101感染细胞株后,仅在p53突变的IGF2 LOI细胞(HT-29)中出现E1A mRNA和蛋白表达,72 h后10 pfu/cell感染的HT-29细胞增殖活率下降(59.4±2.4)%,凋亡率为(38.6±3.2)%；(4)多点注射PBS、Ad312-EGFP、H101和Ad312-E1A治疗HT-29细胞种植的裸鼠移植瘤,结果显示腺病毒H101和Ad312-E1A均能有效的抑制肿瘤的生长并改善其预后。结论：(1)本研究成功的构建了携带IGF2 Imprinting系统和腺病毒复制基因E1A的重组腺病毒；(2)重组腺病毒Ad312-E1A在体内外均能有效的杀伤抑制IGF2 LOI的人结直肠癌细胞；(3) Ad312-E1A可以作为靶向治疗CRC的一个新途径,从而为CRC的基因靶向治疗提供新思路。
[Abstract]:Aim: to construct recombinant adenovirus carrying insulin-like growth factor 2 blotting (IGF2 Imprinting) system and to explore the feasibility of targeting human colorectal cancer (colorectal cancer, CRC) cell line. Methods: (1) PCR was used to amplify human IGF2 Imprinting related structural elements, including H19 promoter enhancer and CTCF fragments, and cloned into pDC-312 vector to construct IGF2Imprinting system; (2) E1A and EGFP fragments were amplified from plasmids TopK and pEGFP-C1 vector, respectively. It was inserted into the recombinant plasmid containing IGF2 Imprinting system to construct the recombinant adenovirus shuttle plasmid. (3) Topl0 competent cells were amplified and purified. Adenovirus shuttle plasmid pDC312-E1A and skeleton plasmid Ad5 were co-transfected into human embryonic kidney cells (HEK293) by liposome Lipo2000. Ad-H19-CTCF-enhancer-E1A and Ad-H19-CTCF-enhancer-EGFP, named Ad312-E1A and Ad312-EGFP respectively; (4) Colorectal cancer cells HT-29 and HCT-8 infected with IGF2 imprinting loss (IGF2 LOI) and SW480 with IGF2 imprinted normal (IGF2 MOI) by adenovirus Ad312-EGFP, and normal colorectal cancer cells with IGF2 blotting (IGF2 MOI), respectively. The expression of EGFP was observed by fluorescence microscope in human gastric mucosal epithelial cell line GES-1. (5) after Ad312-E1A and H101 were infected with different cell lines 48 h later, RT-PCR and Western Blot were used to detect the expression of E1A nRNA and its protein at transcription and translation levels, respectively. CCK-8 assay and flow cytometry were used to detect the tumor killing effect of adenovirus Ad312-E1A in vitro. (6) A nude mouse model of axillary tumor transplantation was established to verify the killing effect of Ad312-E1A in vivo. The results were as follows: (1) the shuttle plasmid pDC312-E1A and the skeleton plasmid Ad5 were successfully transfected into the HEK293 cells at 48h, and the expression of pDC312-E1A was the strongest at 48h, and the cytopathic effect was positive. The suitable titer (1 脳 10 ~ 9 pfu/ml); (_ 2) was obtained by the amplification of three rotaviruses. The recombinant adenovirus Ad312-EGFP was used to infect different cell lines for 48 h to observe the obvious fluorescence of IGF2 LOI cell line (HT-29, HCT-8). (3) mRNA and protein of E1A were only expressed in IGF2 LOI cells (HT-29 HCT-8) after 48 h infection with recombinant adenovirus Ad312-E1A. The proliferative activity of IGF2 LOI cells (HT-29 HCT-8) infected with H101 for 10 pfu/cell was decreased by (69.7 卤5.7)% and (65.2 卤7.1), respectively, and the apoptotic rate was (33.4 卤4.1)% and (29.5 卤2.7), respectively, and that of H101 cells infected with adenolysin H101 was (33.4 卤4.1)% and (29.5 卤2.7), respectively. The proliferative activity of HT-29 cells infected with 10 pfu/cell was decreased (59.4 卤2.4) and the apoptotic rate was (38.6 卤3.2). (4) PBSAd312-EGFPFPH101 and Ad312-E1A were injected to treat the transplanted tumor of HT-29 cells in nude mice. The results showed that both adenovirus H101 and Ad312-E1A could effectively inhibit tumor growth and improve prognosis. Conclusion: (1) the recombinant adenovirus carrying IGF2 Imprinting system and adenovirus replication gene E1A was successfully constructed in this study. (2) the recombinant adenovirus Ad312-E1A could effectively kill human colorectal cancer cells which inhibited IGF2 LOI in vivo and in vitro. (3) Ad312-E1A can be used as a new way to target CRC and provide a new idea for gene targeting therapy of CRC.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.34

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