乙型肝炎病毒介导载脂蛋白和NQO1对肝细胞脂肪化的作用及机制研究

发布时间：2018-08-29 14:18

【摘要】：乙型肝炎病毒（Hepatitis B virus，HBV）导致急慢性乙型肝炎，并与肝硬化和肝细胞癌（Hepatocelluar carcinoma，HCC）的发生发展密切相关。近年来由于脂肪性肝病（Fatty liver disease，FLD）的发病率逐年增加，导致脂肪肝与慢性乙型肝炎（chronic hepatitis B，CHB）合并存在的病例明显增多，对两者相关性的研究逐渐受到重视。但是迄今为止，HBV与脂肪肝的关系尚未完全阐明。本课题组先前通过蛋白质组学和MALDI-TOF/TOF MS技术研究发现HBV影响肝细胞载脂蛋白（Apolipoprotein，Apo）的表达，并降低NAD(P)H醌氧化还原酶1（NAD(P)H dehydrogenase:quinone1，NQO1）的表达，现有研究表明上述因子参与脂肪酸的合成和代谢，对肝脏脂质代谢动态循环平衡的维持起着重要的作用。本研究在此基础上，探讨HBV编码蛋白（LHBs、MHBs、HBs、HBc、HBe、HBpol、HBx）与特定宿主细胞中载脂蛋白表达的对应关系，进一步探讨HBV影响NQO1致肝细胞脂肪化的机制，有助于更深入了解HBV的致病机制，为有效地进行脂肪肝的防治提供理论基础。 本研究第一部分旨在探讨HBV整体水平对Apo基因的影响。通过对HBV细胞株和pRep-HBV质粒瞬时转染细胞中HBsAg及HBeAg的检测，，确定HBV基因组的正常复制和表达；通过realtime RT-PCR的方法，检测了16种Apo基因在HepG2.2.15及HepG2细胞中表达的差异，并进一步用瞬时转染的方法在Huh7、HepG2和Hep3B细胞进行验证，结果表明ApoAI，ApoAII，ApoAV，ApoB，ApoCIII，ApoE，ApoF，ApoH，ApoJ，ApoL1和ApoM和对照组相比转录水平均明显降低，APOD的转录水平和对照组相比明显升高；通过检测不同时间点HBV表达量对上述Apo基因转录水平的影响，证实HBV表达量对Apo基因的表达影响具有时间效应。 本研究第二部分应用AdEasy XL System构建HBV七种编码蛋白的重组腺病毒表达载体Ad-LHBs、Ad-MHBs、Ad-HBs、Ad-HBc、Ad-HBe、Ad-HBpol、Ad-HBx及对照空病毒Ad-GFP，探讨HBV编码蛋白对特定宿主细胞中Apo基因表达的对应关系。通过对腺病毒包装过程的监测和Westernblot检测，成功构建可分别表达HBV LS、MS、S、Core、E、Pol、 X蛋白的重组腺病毒；通过realtime RT-PCR检测HBV七种编码蛋白对Apo基因的表达影响，我们发现HBs下调ApoAII的转录水平，MHBs下调ApoF的转录水平，HBpol下调ApoH的转录水平，且均与HBV整体水平结果一致；通过western blot进一步检测ApoAII、ApoF和ApoH基因蛋白水平的变化，发现HBs下调ApoAII蛋白水平，与转录水平一致；MHBs和HBpol分别上调ApoF和ApoH的蛋白水平，与转录水平不一致，我们考虑可能存在转录、转录后水平、翻译和翻译后水平的多种组合调控，具体机制有待进一步的研究。 本研究第三部分旨在探讨HBV特别是HBx抑制NQO1致肝细胞脂肪化的机制。通过对活细胞内源性ROS和超氧化物的检测，证实HBx通过抑制NQO1的表达从而诱发细胞的氧化应激；通过对细胞内抗氧化酶还原性谷胱甘肽GSH的检测，表明过量的ROS产生可能会破坏细胞内的抗氧化防御体系；应用Annexin-V/PI双染法检测细胞凋亡的发生，证实过量的ROS蓄积引起细胞凋亡；同时通过采用CCK-8检测H2O2处理后细胞的生存率，表明HBx抑制NQO1表达使细胞对H2O2引起的细胞毒性反应更敏感；通过线粒体Δψm和ATP水平的检测我们发现NQO1参与了HBx诱发的线粒体损伤作用。
[Abstract]:Hepatitis B virus (HBV) causes acute and chronic hepatitis B and is closely related to the occurrence and development of liver cirrhosis and hepatocellular carcinoma (HCC). However, the relationship between HBV and fatty liver has not been fully elucidated. Previous proteomics and MALDI-TOF/TOF MS studies have shown that HBV affects apolipoprotein (Apo) expression and reduces NAD (P) Hquinone expression in hepatocytes. The expression of NAD (P) H dehydrogenase: quinone 1 (NQO1) has been shown to be involved in the synthesis and metabolism of fatty acids and play an important role in maintaining the dynamic circulation balance of liver lipid metabolism. The corresponding relationship between apolipoprotein expression and the effect of HBV on NQO1-induced hepatic steatosis will be helpful to understand the pathogenesis of HBV and provide theoretical basis for effective prevention and treatment of fatty liver.
The first part of this study was to investigate the effect of the overall level of HBV on apo gene.The normal replication and expression of HBV genome were determined by detecting HBsAg and HBeAg in HBV cell lines and transiently transfected cells with pRep-HBV plasmids.The expression differences of 16 apo genes in HepG2.2.15 and HepG2 cells were detected by realtime RT-PCR. The results showed that apoAI, ApoAII, ApoAV, ApoB, ApoCIII, ApoE, ApoF, ApoH, ApoJ, ApoL1 and ApoM were significantly lower than those of the control group, and the transcriptional level of APOD was significantly higher than that of the control group at different time points. The transcriptional level of Po gene has confirmed that the expression of HBV has a time effect on the expression of Apo gene.
In the second part of this study, AdEasy XL System was used to construct recombinant adenovirus expression vectors Ad-LHBs, Ad-MHBs, Ad-HBs, Ad-HBc, Ad-HBe, Ad-HBpol, Ad-HBx and control empty virus Ad-GFP for HBV-encoded proteins. The relationship between the expression of Apo gene in specific host cells and the expression of HBV-encoded proteins was investigated by monitoring the packaging process of adenovirus and Western blotting. The recombinant adenovirus expressing HBV LS, MS, S, Core, E, Pol and X proteins was successfully constructed by ernblot. The effect of seven HBV coding proteins on apo gene expression was detected by realtime RT-PCR. We found that HBs down-regulated the transcriptional level of ApoAII, MHBs down-regulated the transcriptional level of ApoF, and HBpol down-regulated the transcriptional level of ApoH, and both were in line with the overall level of HBV. The results were consistent; ApoAII, ApoF and ApoH gene protein levels were further detected by Western blot, and HBs down-regulated ApoAII protein levels, consistent with the transcriptional level; MHBs and HBpol up-regulated ApoF and ApoH protein levels, respectively, inconsistent with the transcriptional level, we considered that there may be transcriptional, post-transcriptional, translation and post-translation levels. A variety of combination control mechanisms need further study.
The third part of this study was designed to explore the mechanism of HBV, especially HBx, inhibiting NQO1-induced hepatocyte steatosis. The detection of endogenous ROS and superoxide in living cells confirmed that HBx could induce oxidative stress by inhibiting NQO1 expression, and the detection of antioxidant enzyme glutathione GSH in cells showed that excessive ROS was produced. The results showed that HBx inhibited NQO1 expression and made the cells more sensitive to H2O2-induced cytotoxicity. Detection of mitochondrial m and ATP levels revealed that NQO1 was involved in HBx induced mitochondrial damage.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62;R575

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