發(fā)布時間：2018-07-08 20:26

  本文選題：食管癌 + Eca109細胞　； 參考：《石河子大學》2017年碩士論文

【摘要】：目的:探討HPV16-E6基因?qū)ca109和Eca9706食管癌細胞生物學特性的影響,為揭示HPV對食管癌細胞的作用機制提供實驗依據(jù)。方法:使用陽離子脂質(zhì)體Lip2000將HPV16-E6基因?qū)隕ca109型和Eca9706型食管癌細胞中,通過計數(shù)熒光細胞占所有細胞的比例來評估轉(zhuǎn)染效率;使用中心復合實驗設(shè)計,實現(xiàn)轉(zhuǎn)染條件的優(yōu)化,使HPV16-E6基因在食管癌細胞中高表達。在分子生物學實驗中,通過RT-PCR來檢測細胞內(nèi)含有目的基因;通過免疫熒光蛋白對目的蛋白在細胞中的表達進行定位;通過western-blot對E6蛋白的表達進行定量分析。每組間具有顯著統(tǒng)計學差異時(p0.01),行細胞生物學實驗。在細胞生物學實驗中,利用CCK-8試劑檢測食管癌細胞的增殖能力變化;通過平板克隆實驗檢測食管癌細胞的集落形成能力變化;通過細胞劃痕實驗檢測食管癌細胞的遷移能力變化,通過Transwell侵襲實驗檢測食管癌細胞的侵襲能力變化。結(jié)果:在分子生物學實驗中:PT-RCR實驗結(jié)果顯示,在Eca109和Eca9706食管癌細胞中,目的基因組的m RNA表達量明顯高于陰性對照組和空白對照組;免疫熒光蛋白實驗結(jié)果顯示,在目的基因組中這兩種食管癌細胞的胞核和胞質(zhì)中,均發(fā)現(xiàn)HPV16-E6基因的表達跡象;Wstern-Blot實驗結(jié)果顯示,在兩種食管癌細胞的目的基因組中,均在17kb處發(fā)現(xiàn)一條明顯的著色帶,因此E6蛋白均有表達。在細胞生物學實驗中:CCK-8檢測結(jié)果表明,轉(zhuǎn)染目的基因的食管癌細胞增殖速率明顯高于陰性對照對照組;平板克隆實驗顯示,目的基因組形成克隆團數(shù)明顯高于空載組,并且目的基因組的細胞團更大;細胞劃痕實驗結(jié)果顯示,與陰性對照組相比,HPV16-E6基因可明顯增加食管癌細胞的遷移能力;Transwell侵襲實驗結(jié)果顯示,攜帶目的基因的食管癌細胞的侵襲能力明顯增強。結(jié)論:成功將HPV16-E6基因整合到Eca109和Eca9706型細胞株并且E6蛋白均可以高表達,證實HPV16-E6可增強食管癌的增殖、遷移和侵襲能力,可能在食管癌的發(fā)生、發(fā)展中起著重要作用。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

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