HPV16E6基因对食管鳞癌细胞增殖、迁移及侵袭的影响

发布时间：2018-07-08 20:26

本文选题：食管癌 + Eca109细胞　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:探讨HPV16-E6基因对Eca109和Eca9706食管癌细胞生物学特性的影响,为揭示HPV对食管癌细胞的作用机制提供实验依据。方法:使用阳离子脂质体Lip2000将HPV16-E6基因导入Eca109型和Eca9706型食管癌细胞中,通过计数荧光细胞占所有细胞的比例来评估转染效率;使用中心复合实验设计,实现转染条件的优化,使HPV16-E6基因在食管癌细胞中高表达。在分子生物学实验中,通过RT-PCR来检测细胞内含有目的基因;通过免疫荧光蛋白对目的蛋白在细胞中的表达进行定位;通过western-blot对E6蛋白的表达进行定量分析。每组间具有显著统计学差异时(p0.01),行细胞生物学实验。在细胞生物学实验中,利用CCK-8试剂检测食管癌细胞的增殖能力变化;通过平板克隆实验检测食管癌细胞的集落形成能力变化;通过细胞划痕实验检测食管癌细胞的迁移能力变化,通过Transwell侵袭实验检测食管癌细胞的侵袭能力变化。结果:在分子生物学实验中:PT-RCR实验结果显示,在Eca109和Eca9706食管癌细胞中,目的基因组的m RNA表达量明显高于阴性对照组和空白对照组;免疫荧光蛋白实验结果显示,在目的基因组中这两种食管癌细胞的胞核和胞质中,均发现HPV16-E6基因的表达迹象;Wstern-Blot实验结果显示,在两种食管癌细胞的目的基因组中,均在17kb处发现一条明显的着色带,因此E6蛋白均有表达。在细胞生物学实验中:CCK-8检测结果表明,转染目的基因的食管癌细胞增殖速率明显高于阴性对照对照组;平板克隆实验显示,目的基因组形成克隆团数明显高于空载组,并且目的基因组的细胞团更大;细胞划痕实验结果显示,与阴性对照组相比,HPV16-E6基因可明显增加食管癌细胞的迁移能力;Transwell侵袭实验结果显示,携带目的基因的食管癌细胞的侵袭能力明显增强。结论:成功将HPV16-E6基因整合到Eca109和Eca9706型细胞株并且E6蛋白均可以高表达,证实HPV16-E6可增强食管癌的增殖、迁移和侵袭能力,可能在食管癌的发生、发展中起着重要作用。
[Abstract]:Objective: to investigate the effect of HPV16-E6 gene on the biological characteristics of Eca109 and Eca9706 esophageal cancer cells, and to provide experimental evidence for revealing the mechanism of HPV acting on esophageal cancer cells. Methods: HPV16-E6 gene was introduced into Eca109 and Eca9706 esophageal cancer cells using cationic liposome Lip2000. The transfection efficiency was evaluated by counting the proportion of fluorescent cells to all the cells, and the transfection conditions were optimized by using the central composite experimental design. HPV16-E6 gene was overexpressed in esophageal carcinoma cells. In molecular biology experiments, RT-PCR was used to detect the target gene in the cells; immunofluorescence protein was used to localize the expression of the target protein in the cells; and western-blot was used to quantitatively analyze the expression of E6 protein. When there was significant statistical difference between each group (p 0.01), the cell biology experiment was carried out. In the cell biology experiment, the proliferation ability of esophageal cancer cells was detected by CCK-8 reagent, and the colony forming ability of esophageal cancer cells was detected by plate cloning assay. The migration ability of esophageal carcinoma cells was detected by cell scratch assay, and the invasion ability of esophageal carcinoma cells was detected by Transwell invasion assay. Results: in the molecular biology experiment, the results showed that the mRNA expression of the target genome in Eca109 and Eca9706 esophageal cancer cells was significantly higher than that in the negative control group and the blank control group, and the results of immunofluorescence protein assay showed that the expression of mRNA in the target genome was significantly higher than that in the negative control group and the blank control group. The expression of HPV16-E6 gene was found in the nucleus and cytoplasm of the two kinds of esophageal cancer cells in the target genome. Wstern-Blot analysis showed that in the target genome of the two esophageal cancer cells, an obvious ribbon was found at the 17kb site. Therefore, E6 protein was expressed. The results of cell biological assay showed that the proliferation rate of esophageal cancer cells transfected with target gene was significantly higher than that of negative control group, and the number of colony formation in target genome was significantly higher than that in no-load group. The results of cell scratch assay showed that HPV16-E6 gene could significantly increase the migration ability of esophageal cancer cells compared with negative control group and the results of Transwell invasion assay showed that HPV16-E6 gene could increase the migration ability of esophageal cancer cells. The invasive ability of esophageal cancer cells carrying the target gene was significantly enhanced. Conclusion: HPV16-E6 gene was successfully integrated into Eca109 and Eca9706 cell lines and E6 protein was highly expressed. It was confirmed that HPV16-E6 can enhance the proliferation, migration and invasion of esophageal carcinoma, and may play an important role in the occurrence and development of esophageal carcinoma.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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