PHB表达与HBV感染关系及对细胞生理功能影响研究

发布时间：2018-02-16 18:54

  本文关键词： 乙型肝炎病毒 PHB 抗病毒 肝细胞癌　出处：《重庆医科大学》2013年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(hepatitis virus，HBV)感染是全球性的公共卫生问题，据统计，每年约有100万人死于HBV感染，我国是全球HBV感染高发区之一。持续性HBV感染是导致慢性肝脏损伤、肝衰竭、肝纤维化、肝硬化以及原发性肝细胞癌(hepatocellular carcinoma, HCC)的主要原因之一。近年来，虽然在HBV感染的抗病毒和免疫治疗方面取得了一些突破，但仍缺乏根治慢性HBV感染的办法。最大限度的长期抑制HBV复制，减轻肝细胞炎症坏死及肝纤维化，延缓或减少肝脏失代偿、肝硬化、HCC和并发症的发生，从而改善生活质量和延长生存时间是当前HBV感染治疗的总体目标。Prohibitin(PHB)是广泛存在于各种生物组织细胞中具有明显抗细胞增殖、抗肿瘤作用的蛋白分子。PHB蛋白作为一种新型的分子伴侣主要分布于细胞膜、线粒体内膜及细胞核中，参与细胞增殖、凋亡、转录、代谢、衰老、细胞稳态及线粒体蛋白质折叠等重要细胞生物学功能的调节，参与了肿瘤、衰老及退行性病变、糖尿病、肥胖、炎症性肠病等疾病的发生发展过程。iTRAQ技术（同位素相对标记和绝对定量技术）是近年来最新研发的新的蛋白组学定量研究技术，已在肝脏疾病的研究中广泛用。在前期研究中，我们应用此技术对正常人肝组织和慢乙肝患者肝组织进行了蛋白组学分析，发现PHB蛋白在慢乙肝患者肝组织表达较正常肝组织降低。为此，本课题旨在通过用抗病毒药物干预HBV复制、转染PHB质粒等方法，运用Realtime PCR（RT-PCR）、Westernblotting及质粒转染等技术，研究PHB表达和HBV感染的关系以及PHB对人肝癌细胞HepG2.2.15生物学功能的影响，，探索PHB在HBV感染组织和细胞表达降低的分子机制和生理意义。 在研究中，首先我们运用免疫组化、RT-PCR、Western blottting等技术分别对乙肝患者肝组织和HepG2.2.15细胞株进行PHB基因转录和蛋白质表达水平的验证，结果发现乙肝患者肝组织和HepG2.2.15细胞株中PHB mRNA和蛋白质表达分别明显较正常肝组织和HepG2细胞株明显降低，提示HBV感染可能下调PHB表达；为进一步研究HBV复制对PHB表达的影响，在HepG2.2.15细胞培养液中加入抗病毒药物（拉米夫定和替诺福韦），观察HBV复制被抑制后PHB表达情况，结果PHB mRNA和蛋白质表达均上调；为研究HBV感染引起PHB表达下调的具体机制，我们在HepG2细胞株上分段转染HBV编码蛋白的基因质粒（PCMV-tag2B-HBss、PCMV-tag2B-HBe、PCMV-tag2B-HBc、 PCMV-tag2B-HBx、 PCMV-tag2B-HBls andPCMV-tag2B-HBms），结果发现HepG2细胞株在转染HBx蛋白基因质粒（PCMV-tag2B-HBx）后PHB蛋白表达显著增加；为观察PHB表达水平改变对细胞生理功能的影响，我们对HepG2.2.15细胞转染PHB质粒（PCMV6-AC-GFP-PHB），观察其细胞周期、增殖、凋亡等生理功能变化，结果HepG2.2.15细胞周期被阻滞于G1/S期、细胞增殖被抑制、细胞凋亡增加；最后，用Westernblotting等技术研究慢乙肝病人有效抗病毒前后肝组织PHB表达的变化，结果发现有效抗病毒HBV-DNA转阴后肝组织PHB蛋白表达明显高于治疗前。 本课题研究结果表明，HBV感染是慢性乙型肝炎肝组织和HepG2.2.15细胞株PHB表达降低的原因之一，PHB在肝细胞中发挥着抗细胞增殖和促细胞凋亡的作用，有效抗病毒治疗可以增加肝细胞内PHB表达水平，从而为HBV感染抗病毒治疗的必要性及HBV感染后对细胞生理功能的影响和肝癌的发生发展作用提供理论基础，为治疗肝癌的药物靶点研究提供了重要科学依据，PHB有成为肝癌的诊断和病情判断指标的潜在可能。
[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),缁撴灉鍙戠幇HepG2缁嗚優鏍�鍦ㄨ浆鏌揌Bx铔嬬櫧鍩哄洜璐ㄧ矑(PCMV-tag2B-HBx)鍚嶱HB铔嬬櫧琛ㄨ揪鏄捐憲澧炲姞锛涗负瑙傚療PHB琛ㄨ揪姘村钩鏀瑰彉瀵圭粏鑳炵敓鐞嗗姛鑳界殑褰卞搷,鎴戜滑瀵笻epG2.2.15缁嗚優杞�鏌揚HB璐ㄧ矑(PCMV6-AC-GFP-PHB),瑙傚療鍏剁粏鑳炲懆鏈

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