重组溶瘤腺病毒RCAdC68.pⅨ-EGFP的构建及其对结肠癌细胞增殖的影响

发布时间：2018-03-30 13:53

本文选题：溶瘤腺病毒　切入点：等温组装　出处：《苏州大学》2015年硕士论文

【摘要】：目的本实验通过等温组装法构建重组溶瘤腺病毒RCAd C68.p IX-EGFP,并通过比较RCAd C68.p IX-EGFP与复制缺陷型重组腺病毒Ad C68-ΔE1-EGFP对结肠癌细胞的杀伤作用,初步观察RCAd C68.p IX-EGFP对结肠癌细胞增殖的影响。方法以黑猩猩型腺病毒Ad C68为模板,利用等温组装法构建腺病毒衣壳蛋白p IX展示EGFP的重组溶瘤腺病毒质粒,HEK293细胞中包装成完整病毒并扩增、纯化,获得具有溶瘤效应可表达EGFP的重组溶瘤腺病毒载体,命名为RCAd C68.p IX-EGFP。以不同的病毒剂量(100、500、2500、12500vp/cell)感染结肠癌细胞HCT 116、NCI-H508、HT-29及人胚肾细胞HEK-293,分别于感染后第3、5、7天用MTT法检测RCAd C68.p IX-EGFP对不同细胞的抑制率,结晶紫染色实验检测RCAd C68.p IX-EGFP对不同细胞的杀伤作用,荧光显微镜与相差显微镜观察细胞形态学变化和病变情况。结果重组溶瘤腺病毒载体RCAd C68.p IX-EGFP构建成功。RCAd C68.p IX-EGFP对结肠癌细胞HCT116、NCI-H508、HT-29的增殖抑制作用与病毒感染剂量及感染时间成正比,以500vp/cell病毒感染剂量感染细胞7天后,RCAd C68.p IX-EGFP对HCT 116、NCI-H508、HT-29、HEK293细胞的抑制率分别为86.70%、96.85%、99.70%、99.99%,Ad C68-ΔE1-EGFP的细胞抑制率分别为6.57%、11.46%、9.11%、99.99%,RCAd C68.p IX-EGFP与Ad C68-ΔE1-EGFP对结肠细胞增殖抑制作用的差异有统计学意义(P0.05),而对HEK-293细胞增殖抑制作用的差异无统计学意义(P0.05)。结论本研究成功构建重组溶瘤腺病毒载体RCAd C68.p IX-EGFP,RCAd C68.p IX-EGFP能显著抑制结肠癌细胞HCT 116、NCI-H508、HT-29的增殖,初步显示出RCAd C68.p IX-EGFP作为溶瘤病毒载体治疗肿瘤的潜能。
[Abstract]:Objective to construct recombinant adenovirus RCAd C68.p IX-EGFP by isothermal assembly, and to compare the cytotoxicity of RCAd C68.p IX-EGFP and replication-deficient recombinant adenovirus Ad C68- 螖 E1-EGFP on colon cancer cells. The effect of RCAd C68.p IX-EGFP on the proliferation of colon cancer cells was studied. Methods the chimpanzee adenovirus Ad C68 was used as a template. Adenovirus capsid protein (PIX) was constructed by isothermal assembly method. The recombinant adenovirus plasmid pIX, which displayed EGFP, was packaged into a complete virus, amplified and purified, and a recombinant adenovirus vector expressing EGFP was obtained. It was named RCAd C68.p IX-EGFP.Colon cancer cell line HCT 116nCI-H508 HT-29 and human embryonic kidney cell line HEK-293were infected with different doses of RCAd C68.p IX-EGFP.The inhibitory rates of RCAd C68.p IX-EGFP on different cells were detected by MTT method on the 3rd day after infection. The cytotoxicity of RCAd C68.p IX-EGFP to different cells was detected by crystal violet staining. Results the recombinant adenovirus vector RCAd C68.p IX-EGFP was successfully constructed to inhibit the proliferation of human colon cancer cell line HCT116NCI-H508H508HT-29. Results the inhibitory effect of recombinant adenovirus vector RCAd C68.p IX-EGFP on the proliferation of human colon cancer cell line HCT116NCI-H508h-29 was in direct proportion to the dose and time of infection. The inhibition rates of RCAd C68.p IX-EGFP on HCT 116NCI-H508h-29HEK293 cells were 86.70 and 96.96.858.70- 螖 E1-EGFP respectively after 7 days of 500vp/cell virus infection. The inhibitory rates of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP on the proliferation of colon cells were significantly different from those of Ad C68- 螖 E1-EGFP, respectively (6.5711.469.115p), while the inhibitory rates of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP on the proliferation of colon cells were significantly different from those of RCAd C68.p IX-EGFP and Ad C68- 螖 E1-EGFP. Conclusion the recombinant adenovirus vector RCAd C68.p IX-EGFPGFP RCAd C68.p IX-EGFP can significantly inhibit the proliferation of colon cancer cell line HCT 116NCI-H508H508h-29, and there is no significant difference in the inhibition of proliferation of HEK-293 cells by P0.055.Conclusion the recombinant adenovirus vector RCAd C68.p IX-EGFPFP-RCAd C68.p IX-EGFP can significantly inhibit the proliferation of colon cancer cells. The potential of RCAd C 68 p IX-EGFP to treat tumor as a vector of oncolytic virus was preliminarily demonstrated.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.3

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