本文選題：食管癌 + Eca109細(xì)胞��； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:探討HPV16-E6基因?qū)ca109和Eca9706食管癌細(xì)胞生物學(xué)特性的影響,為揭示HPV對(duì)食管癌細(xì)胞的作用機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:使用陽(yáng)離子脂質(zhì)體Lip2000將HPV16-E6基因?qū)隕ca109型和Eca9706型食管癌細(xì)胞中,通過(guò)計(jì)數(shù)熒光細(xì)胞占所有細(xì)胞的比例來(lái)評(píng)估轉(zhuǎn)染效率;使用中心復(fù)合實(shí)驗(yàn)設(shè)計(jì),實(shí)現(xiàn)轉(zhuǎn)染條件的優(yōu)化,使HPV16-E6基因在食管癌細(xì)胞中高表達(dá)。在分子生物學(xué)實(shí)驗(yàn)中,通過(guò)RT-PCR來(lái)檢測(cè)細(xì)胞內(nèi)含有目的基因;通過(guò)免疫熒光蛋白對(duì)目的蛋白在細(xì)胞中的表達(dá)進(jìn)行定位;通過(guò)western-blot對(duì)E6蛋白的表達(dá)進(jìn)行定量分析。每組間具有顯著統(tǒng)計(jì)學(xué)差異時(shí)(p0.01),行細(xì)胞生物學(xué)實(shí)驗(yàn)。在細(xì)胞生物學(xué)實(shí)驗(yàn)中,利用CCK-8試劑檢測(cè)食管癌細(xì)胞的增殖能力變化;通過(guò)平板克隆實(shí)驗(yàn)檢測(cè)食管癌細(xì)胞的集落形成能力變化;通過(guò)細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)食管癌細(xì)胞的遷移能力變化,通過(guò)Transwell侵襲實(shí)驗(yàn)檢測(cè)食管癌細(xì)胞的侵襲能力變化。結(jié)果:在分子生物學(xué)實(shí)驗(yàn)中:PT-RCR實(shí)驗(yàn)結(jié)果顯示,在Eca109和Eca9706食管癌細(xì)胞中,目的基因組的m RNA表達(dá)量明顯高于陰性對(duì)照組和空白對(duì)照組;免疫熒光蛋白實(shí)驗(yàn)結(jié)果顯示,在目的基因組中這兩種食管癌細(xì)胞的胞核和胞質(zhì)中,均發(fā)現(xiàn)HPV16-E6基因的表達(dá)跡象;Wstern-Blot實(shí)驗(yàn)結(jié)果顯示,在兩種食管癌細(xì)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(dá)。在細(xì)胞生物學(xué)實(shí)驗(yàn)中:CCK-8檢測(cè)結(jié)果表明,轉(zhuǎn)染目的基因的食管癌細(xì)胞增殖速率明顯高于陰性對(duì)照對(duì)照組;平板克隆實(shí)驗(yàn)顯示,目的基因組形成克隆團(tuán)數(shù)明顯高于空載組,并且目的基因組的細(xì)胞團(tuán)更大;細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,與陰性對(duì)照組相比,HPV16-E6基因可明顯增加食管癌細(xì)胞的遷移能力;Transwell侵襲實(shí)驗(yàn)結(jié)果顯示,攜帶目的基因的食管癌細(xì)胞的侵襲能力明顯增強(qiáng)。結(jié)論:成功將HPV16-E6基因整合到Eca109和Eca9706型細(xì)胞株并且E6蛋白均可以高表達(dá),證實(shí)HPV16-E6可增強(qiáng)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(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王平;劉珊;程波;吳西釗;武迪;許琳;石建玲;段戀;孫鎖柱;;siRNA介導(dǎo)的HPV16E6/E7基因沉默對(duì)宮頸癌SiHa增殖及凋亡的影響[J];診斷病理學(xué)雜志;2015年12期

2 嚴(yán)愛(ài)芬;劉婉霞;劉芳;劉靖;張雅潔;唐冬生;;TALEN質(zhì)粒轉(zhuǎn)染HEK-293T細(xì)胞的條件優(yōu)化[J];基因組學(xué)與應(yīng)用生物學(xué);2015年12期

3 白彩云;王延明;;宮頸上皮內(nèi)瘤變患者高危型HPV感染與潛在惡性生物學(xué)行為的關(guān)系[J];海南醫(yī)學(xué)院學(xué)報(bào);2015年09期

4 王莉;黨裔武;莫小亮;陳罡;韋康來(lái);羅殿中;;宮頸病變中hTERC基因擴(kuò)增與hTERT蛋白表達(dá)的關(guān)系及意義[J];診斷病理學(xué)雜志;2014年09期

5 陳衛(wèi)剛;楊春梅;徐麗紅;張寧;劉曉燕;馬云貴;霍曉玲;韓玉勝;田德安;鄭勇;;Gene Chip Technology Used in the Detection of HPV Infection in Esophageal Cancer of Kazakh Chinese in Xinjiang Province[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2014年03期

6 林克榮;陳國(guó)榮;王維維;林朗;黃兆浪;鄭向陽(yáng);丁振計(jì);;食道乳頭狀瘤與人乳頭狀瘤病毒感染的相關(guān)性研究[J];中華醫(yī)院感染學(xué)雜志;2014年03期

7 朱紅霞;周曉波;劉梅;全蘭平;徐寧志;;HPV16 E7蛋白對(duì)TNF-α誘導(dǎo)食管癌細(xì)胞凋亡及其機(jī)制的探討[J];中華腫瘤防治雜志;2013年01期

8 周艷芳;陳曉愛(ài);葉美紅;帥心濤;鄧宇;;正交設(shè)計(jì)優(yōu)化聚乙烯亞胺介導(dǎo)肝癌細(xì)胞基因轉(zhuǎn)染效率的研究[J];生物醫(yī)學(xué)工程學(xué)雜志;2011年01期

9 顧立怡;賽麗曼;候小麗;陳剛;李鋒;劉春霞;陳云昭;常彬;楊蘭;梁偉華;胡建明;;HPV16感染與新疆哈薩克族食管癌發(fā)病及臨床病理學(xué)研究[J];中國(guó)癌癥雜志;2010年09期

10 江黎珠;呂彩鳳;盧虹伊;陳鴻雁;;人乳頭瘤病毒16E6蛋白、細(xì)胞周期素D_1及端粒酶逆轉(zhuǎn)錄酶在鼻咽癌組織中的表達(dá)及意義[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2009年12期

相關(guān)碩士學(xué)位論文 前1條

1 董紅超;多重PCR-質(zhì)譜技術(shù)檢測(cè)新疆哈薩克族食管癌HPV感染及其基因型分布[D];石河子大學(xué);2014年

，

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