本文關(guān)鍵詞： 肝癌 SIRT6 凋亡 Bax　出處：《重慶醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：通過研究SIRT6調(diào)控Bax凋亡信號(hào)通路促進(jìn)肝癌細(xì)胞凋亡逃逸,來探討肝細(xì)胞肝癌的發(fā)生發(fā)展機(jī)制。方法：1.利用Western Blot和qRT-PCR檢測SIRT6在101例肝癌病人和各種肝癌細(xì)胞系中的表達(dá)。Kaplan-Meier分析SIRT6與肝癌病人預(yù)后相關(guān)。2.利用慢病毒介導(dǎo)技術(shù)在肝癌細(xì)胞上沉默SIRT6基因的表達(dá),WesternBlot驗(yàn)證SIRT6沉默效率；通過臺(tái)盼藍(lán)染色實(shí)驗(yàn)分析SIRT6基因沉默對肝癌細(xì)胞增殖的影響；平板集落實(shí)驗(yàn)檢測SIRT6基因沉默對肝癌細(xì)胞集落形成能力的影響；EdU標(biāo)記實(shí)驗(yàn)檢測SIRT6基因沉默對肝癌細(xì)胞DNA合成的影響；流式細(xì)胞計(jì)數(shù)和Western Blot檢測SIRT6基因沉默對細(xì)胞凋亡的影響。3.在永生化的肝細(xì)胞MIHA上過表達(dá)SIRT6, WesternBlot驗(yàn)證SIRT6過表達(dá)是否成功；通過臺(tái)盼藍(lán)染色實(shí)驗(yàn)分析SIRT6對永生化肝細(xì)胞增殖的影響；軟瓊脂集落形成實(shí)驗(yàn)檢測SIRT6對永生化肝細(xì)胞非錨定生長能力的影響；EdU標(biāo)記實(shí)驗(yàn)檢測SIRT6對永生化肝細(xì)胞DNA合成的影響；流式細(xì)胞計(jì)數(shù)和Western Blot檢測SIRT6對永生化肝細(xì)胞凋亡的影響。4.利用qRT-PCR分析SIRT6沉默后肝癌細(xì)胞內(nèi)凋亡相關(guān)基因mRNA水平的變化；繼而使用Western Blot驗(yàn)證篩選出的凋亡相關(guān)蛋白的變化水平。利用免疫細(xì)胞化學(xué)和Western Blot分析Bax蛋白在SIRT6基因沉默后細(xì)胞定位的變化；利用臺(tái)盼藍(lán)染色實(shí)驗(yàn)檢測沉默Bax基因?qū)IRT6基因沉默引起的肝癌細(xì)胞增殖的影響；利用流式細(xì)胞學(xué)和Western Blot檢測沉默Bax對SIRT6基因沉默引起的肝癌細(xì)胞凋亡的影響。5.利用雙熒光素酶報(bào)告系統(tǒng)分析Bax啟動(dòng)子的關(guān)鍵區(qū)域,并檢測SIRT6基因沉默對Bax基因啟動(dòng)子的關(guān)鍵區(qū)域的影響；ChIP檢測SIRT6對Bax啟動(dòng)子的關(guān)鍵區(qū)域組蛋白乙�；稽c(diǎn)的影響；ChIP進(jìn)一步檢測SIRT6基因沉默對Bax,啟動(dòng)子轉(zhuǎn)錄因子的影響。6.利用Western Blot檢測SIRT6和Ku70在細(xì)胞質(zhì)和細(xì)胞核中的定位；IP實(shí)驗(yàn)檢測SIRT6與Ku70的相互關(guān)系。Western Blot檢測缺失SIRT6對Ku70蛋白表達(dá)量的影響。IP實(shí)驗(yàn)檢測SIRT6對Ku70乙�；降挠绊�；IP實(shí)驗(yàn)進(jìn)一步檢測SIRT6基因沉默對Ku70-Bax復(fù)合物的影響。7.在在荷氏移植瘤模型試驗(yàn)中驗(yàn)證SIRT6對肝癌細(xì)胞生長的影響。結(jié)果：1.在本研究中我們發(fā)現(xiàn)SIRT6在大量肝癌病人中表達(dá)明顯增高,并且SIRT6增高與肝細(xì)胞肝癌病人不良預(yù)后(p=0.024)高度相關(guān)。同時(shí)發(fā)現(xiàn)SIRT6在肝癌細(xì)胞系上表達(dá)水平較永生化肝細(xì)胞MIHA和原代肝細(xì)胞PHH高。2.在多個(gè)肝癌細(xì)胞系上沉默SIRT6可以抑制肝癌細(xì)胞的增殖,并抑制肝癌細(xì)胞DNA合成和抑制肝癌細(xì)胞集落形成；沉默SIRT6可以誘導(dǎo)肝癌細(xì)胞凋亡。3.在永生化肝細(xì)胞上過表達(dá)SIRT6可以促進(jìn)永生化肝細(xì)胞增殖,促進(jìn)永生化肝細(xì)胞DNA合成和促進(jìn)永生化肝細(xì)胞非錨定生長能力；SIRT6可以降低阿霉素誘導(dǎo)的凋亡。4.SIRT6可以通過Bax信號(hào)通路來發(fā)揮抗肝癌細(xì)胞凋亡作用。SIRT6缺失可以促進(jìn)Bax從細(xì)胞核和細(xì)胞質(zhì)向線粒體轉(zhuǎn)位。沉默Bax可以拮抗SIRT6缺失引起的肝癌細(xì)胞凋亡。5.SIRT6被招募到Bax啟動(dòng)子上,通過去乙�；M蛋白H3K9位點(diǎn),抑制Bax啟動(dòng)子活性。在缺失SIRT6時(shí),結(jié)合于Bax啟動(dòng)子上的轉(zhuǎn)錄因子p53和E2F-1明顯增加。6.SIRT6、Bax和Ku70可以在細(xì)胞質(zhì)中形成復(fù)合物,缺失SIRT6可以通過乙酰化Ku70促進(jìn)Ku70-Bax復(fù)合物的分離,阻止Bax向線粒體轉(zhuǎn)位。7.在荷氏移植瘤模型試驗(yàn)中發(fā)現(xiàn)SIRT6基因沉默可以抑制移植瘤的生長。結(jié)論：SIRT6在肝細(xì)胞肝癌形成中起著促進(jìn)進(jìn)用,因此針對SIRT6的治療可能成為肝細(xì)胞肝癌治療的一個(gè)靶點(diǎn)。
[Abstract]:Objective: through the cell apoptosis of SIRT6 on regulating the apoptosis of Bax signaling pathway to escape, to explore the mechanism of the occurrence and development of hepatocellular carcinoma. Methods: the expression of.Kaplan-Meier by Western Blot and qRT-PCR 1. SIRT6 was detected in 101 patients with hepatocellular carcinoma and hepatocellular carcinoma cell lines of SIRT6 and.2. in patients with hepatocellular carcinoma prognosis by lentivirus mediated expression of SIRT6 gene silencing in human hepatocellular carcinoma cells, WesternBlot verification of SIRT6 silencing efficiency; through the analysis of the influence of SIRT6 gene silencing by trypan blue staining on hepatocellular carcinoma cell proliferation experiment; colony assay of SIRT6 gene silencing colony forming ability of hepatoma cells; the effect of silencing SIRT6 gene detection EdU labeling experiments on the synthesis of DNA cells; gene silencing of SIRT6 detected by flow cytometry and Western effect of Blot on apoptosis of.3. in immortalized hepatocytes The expression of SIRT6 cell MIHA, WesternBlot verification SIRT6 overexpression was successful; trypan through analysis on the effect of SIRT6 on immortalized hepatocyte proliferation blue staining experiment; soft agar assay of SIRT6 immortalized hepatocyte non anchored growth ability; EdU marker assays of SIRT6 liver cells DNA the synthesis of eternal life; influence of flow cytometry and Western Blot detected SIRT6 of immortalized hepatocytes apoptosis of hepatoma cells by qRT-PCR.4. analysis of apoptosis related gene mRNA levels after SIRT6 silencing; changes in the level and then use Western Blot to verify the screened protein related apoptosis. Changes of Bax protein gene in SIRT6 cells silence after positioning by using immunocytochemistry and Western Blot; using trypan blue staining of hepatocellular carcinoma cells Bax gene silencing assay on SIRT6 induced gene silencing. 孌栫殑褰卞搷錛涘埄鐢ㄦ祦寮忕粏鑳?yōu)瀛﹀拰W(xué)estern Blot媯，

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