發(fā)布時間：2018-02-16 18:54

  本文關(guān)鍵詞： 乙型肝炎病毒 PHB 抗病毒 肝細(xì)胞癌　出處：《重慶醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：乙型肝炎病毒(hepatitis virus，HBV)感染是全球性的公共衛(wèi)生問題，據(jù)統(tǒng)計，每年約有100萬人死于HBV感染，我國是全球HBV感染高發(fā)區(qū)之一。持續(xù)性HBV感染是導(dǎo)致慢性肝臟損傷、肝衰竭、肝纖維化、肝硬化以及原發(fā)性肝細(xì)胞癌(hepatocellular carcinoma, HCC)的主要原因之一。近年來，雖然在HBV感染的抗病毒和免疫治療方面取得了一些突破，但仍缺乏根治慢性HBV感染的辦法。最大限度的長期抑制HBV復(fù)制，減輕肝細(xì)胞炎癥壞死及肝纖維化，延緩或減少肝臟失代償、肝硬化、HCC和并發(fā)癥的發(fā)生，從而改善生活質(zhì)量和延長生存時間是當(dāng)前HBV感染治療的總體目標(biāo)。Prohibitin(PHB)是廣泛存在于各種生物組織細(xì)胞中具有明顯抗細(xì)胞增殖、抗腫瘤作用的蛋白分子。PHB蛋白作為一種新型的分子伴侶主要分布于細(xì)胞膜、線粒體內(nèi)膜及細(xì)胞核中，參與細(xì)胞增殖、凋亡、轉(zhuǎn)錄、代謝、衰老、細(xì)胞穩(wěn)態(tài)及線粒體蛋白質(zhì)折疊等重要細(xì)胞生物學(xué)功能的調(diào)節(jié)，參與了腫瘤、衰老及退行性病變、糖尿病、肥胖、炎癥性腸病等疾病的發(fā)生發(fā)展過程。iTRAQ技術(shù)（同位素相對標(biāo)記和絕對定量技術(shù)）是近年來最新研發(fā)的新的蛋白組學(xué)定量研究技術(shù)，已在肝臟疾病的研究中廣泛用。在前期研究中，我們應(yīng)用此技術(shù)對正常人肝組織和慢乙肝患者肝組織進(jìn)行了蛋白組學(xué)分析，發(fā)現(xiàn)PHB蛋白在慢乙肝患者肝組織表達(dá)較正常肝組織降低。為此，本課題旨在通過用抗病毒藥物干預(yù)HBV復(fù)制、轉(zhuǎn)染PHB質(zhì)粒等方法，運用Realtime PCR（RT-PCR）、Westernblotting及質(zhì)粒轉(zhuǎn)染等技術(shù)，研究PHB表達(dá)和HBV感染的關(guān)系以及PHB對人肝癌細(xì)胞HepG2.2.15生物學(xué)功能的影響，，探索PHB在HBV感染組織和細(xì)胞表達(dá)降低的分子機(jī)制和生理意義。 在研究中，首先我們運用免疫組化、RT-PCR、Western blottting等技術(shù)分別對乙肝患者肝組織和HepG2.2.15細(xì)胞株進(jìn)行PHB基因轉(zhuǎn)錄和蛋白質(zhì)表達(dá)水平的驗證，結(jié)果發(fā)現(xiàn)乙肝患者肝組織和HepG2.2.15細(xì)胞株中PHB mRNA和蛋白質(zhì)表達(dá)分別明顯較正常肝組織和HepG2細(xì)胞株明顯降低，提示HBV感染可能下調(diào)PHB表達(dá)；為進(jìn)一步研究HBV復(fù)制對PHB表達(dá)的影響，在HepG2.2.15細(xì)胞培養(yǎng)液中加入抗病毒藥物（拉米夫定和替諾福韋），觀察HBV復(fù)制被抑制后PHB表達(dá)情況，結(jié)果PHB mRNA和蛋白質(zhì)表達(dá)均上調(diào)；為研究HBV感染引起PHB表達(dá)下調(diào)的具體機(jī)制，我們在HepG2細(xì)胞株上分段轉(zhuǎn)染HBV編碼蛋白的基因質(zhì)粒（PCMV-tag2B-HBss、PCMV-tag2B-HBe、PCMV-tag2B-HBc、 PCMV-tag2B-HBx、 PCMV-tag2B-HBls andPCMV-tag2B-HBms），結(jié)果發(fā)現(xiàn)HepG2細(xì)胞株在轉(zhuǎn)染HBx蛋白基因質(zhì)粒（PCMV-tag2B-HBx）后PHB蛋白表達(dá)顯著增加；為觀察PHB表達(dá)水平改變對細(xì)胞生理功能的影響，我們對HepG2.2.15細(xì)胞轉(zhuǎn)染PHB質(zhì)粒（PCMV6-AC-GFP-PHB），觀察其細(xì)胞周期、增殖、凋亡等生理功能變化，結(jié)果HepG2.2.15細(xì)胞周期被阻滯于G1/S期、細(xì)胞增殖被抑制、細(xì)胞凋亡增加；最后，用Westernblotting等技術(shù)研究慢乙肝病人有效抗病毒前后肝組織PHB表達(dá)的變化，結(jié)果發(fā)現(xiàn)有效抗病毒HBV-DNA轉(zhuǎn)陰后肝組織PHB蛋白表達(dá)明顯高于治療前。 本課題研究結(jié)果表明，HBV感染是慢性乙型肝炎肝組織和HepG2.2.15細(xì)胞株P(guān)HB表達(dá)降低的原因之一，PHB在肝細(xì)胞中發(fā)揮著抗細(xì)胞增殖和促細(xì)胞凋亡的作用，有效抗病毒治療可以增加肝細(xì)胞內(nèi)PHB表達(dá)水平，從而為HBV感染抗病毒治療的必要性及HBV感染后對細(xì)胞生理功能的影響和肝癌的發(fā)生發(fā)展作用提供理論基礎(chǔ)，為治療肝癌的藥物靶點研究提供了重要科學(xué)依據(jù)，PHB有成為肝癌的診斷和病情判斷指標(biāo)的潛在可能。
[Abstract]:Hepatitis B virus (hepatitis virus, HBV) infection is a global public health problem, according to statistics, there are about 1 million people died of HBV infection each year, China is one of the world's high incidence of HBV infection. Persistent HBV infection is the leading cause of chronic liver injury, liver failure, liver fibrosis, cirrhosis and hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the main reasons. In recent years, although the antiviral and immune treatment of HBV infection has made some breakthroughs, but still lack of cure of chronic HBV infection. The maximum long-term inhibition of HBV replication, reduce inflammatory necrosis of liver cells and liver fibrosis, delay or reduce liver decompensation cirrhosis HCC and the occurrence of complications, so as to improve the quality of life and prolong the survival time of the overall goal of treatment of.Prohibitin HBV infection (PHB) is widespread in a variety of biological tissues with The effect of anti proliferation, anti tumor effect of protein.PHB protein as a novel molecular chaperones are mainly distributed in the cell membrane, mitochondria and nucleus, involved in cell proliferation, apoptosis, transcription, metabolism, aging, regulating cellular homeostasis and mitochondrial protein folding and other important biological functions in cells, involved in cancer, aging and degenerative disease, diabetes, obesity, disease occurrence and development of.ITRAQ technology such as inflammatory bowel disease (relative and absolute quantification isotope labeled) is a new protein in recent years the latest research and development of quantitative research technology, has been widely used in the study of liver diseases. In previous study, we applied this technique to human normal liver tissues and liver tissues of patients with chronic hepatitis B by proteomic analysis, found that the expression of PHB protein in liver tissue of patients with chronic hepatitis B compared with normal liver tissue decreased. Therefore, the class theme Through the use of antiviral drugs in the intervention of HBV replication, plasmid PHB and other methods, the use of Realtime PCR (RT-PCR), Westernblotting and plasmid transfection technology, research on the influence of PHB expression and HBV infection and PHB on human hepatoma cell biological function of HepG2.2.15, explore PHB in HBV infected cells and tissues expression of molecular mechanism and physiological significance decreased.
In this study, we used immunohistochemistry, RT-PCR, Western blottting respectively. Expression of PHB gene transcription and protein level verification of liver tissues of patients with hepatitis B and HepG2.2.15 cells, results showed that the liver tissue of patients with hepatitis B and HepG2.2.15 cells expressed PHB mRNA and protein respectively was significantly higher than that of normal liver tissue and HepG2 cell line significantly decreased, suggesting that HBV infection may decrease the expression of PHB; for the further study of HBV replication effect on the expression of PHB, in HepG2.2.15 cell culture solution of antiviral drugs (lamivudine and tenofovir), observe HBV replication was inhibited after the expression of PHB, the PHB mRNA and protein expression were up-regulated on HBV infected by PHB; the expression of specific mechanisms of down-regulation of gene transfection of HBV plasmid we segment encoding protein in HepG2 cell line (PCMV-tag2B-HBss, PCMV-tag2B-HBe, PCMV-tag2. B-HBc, PCMV-tag2B-HBx, PCMV-tag2B-HBls andPCMV-tag2B-HBms),緇撴灉鍙戠幇HepG2緇嗚優(yōu)鏍湪杞煋HBx铔嬬櫧鍩哄洜璐ㄧ矑(PCMV-tag2B-HBx)鍚嶱HB铔嬬櫧琛ㄨ揪鏄捐憲澧炲姞錛涗負(fù)瑙傚療PHB琛ㄨ揪姘村鉤鏀瑰彉瀵圭粏鑳?yōu)鐢熺悊鍔熻兘鐨勫奖鍝?鎴戜滑瀵笻epG2.2.15緇嗚優(yōu)杞煋PHB璐ㄧ矑(PCMV6-AC-GFP-PHB),瑙傚療鍏剁粏鑳?yōu)鍛ㄦ?

本文編號：1516205