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
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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。
Infection of human hepatitis B virus (HBV) resulted in acute and chronichepatitis, and closely related to cirrhosis and hepatocellular carcinoma (HCC).Recently, with the increasing prevalence of Nonalcoholic fatty liver disease (NAFLD),hepatic steatosis along with chronic viral hepatitis B in persons become a commonphenomenon. Therefore, studies on the interaction between hepatic steatosis and HBVbecome more and more important. However, the exact association of HBV infectionand hepatic steatosis remains elusive. Previous studies from our laboratory haveshown that the protein level of Apolipoprotein is marked affected and NQO1issignificantly lower by using a proteomic approach and MALDI-TOF/TOF MSanalysis. These proteins may play important roles in lipid metabolism and progressionof hepatic steatosis. And in that basis, we investigated the effect of HBV protein(LHBs、MHBs、HBs、HBc、HBe、HBpol、HBx) on Apolipoprotein (Apo) expression,and further investigated the mechanism of HBx involvement in hepatic steatosis viaNQO1repression. These data may suggested a mechanism for HBV-associatedpathogenesis and provide theoretical basis for the effective prevention and treatmentof fatty liver.
The first part of this study is to investigate whether HBV affected transcriptionApo genes. HBsAg and HBeAg were measured by ELISA kit to confirmed HBVreplication and expression on stable HBV-producing hepatoma cell line and transienttransfection cells. Realtime RT-PCR to examine the16Apo genes transcriptional levelof HepG2.2.15and HepG2cells. And further confirmed by transient transfectionassay. The results showed that the transcriptional level of ApoAI，ApoAII，ApoAV，ApoB，ApoCIII，ApoE，ApoF，ApoH，ApoJ，ApoL1and ApoM is marked lowerin HepG2.2.15cells, while ApoD is significantly higher compared with that in control cell line. Besides, the effect of HBV on Apo genes transcriptional level istime-dependent.
In the second part of this study, AdEasy XL System was used to constructrecombinant adenovirus Ad-LHBs、Ad-MHBs、Ad-HBs、Ad-HBc、Ad-HBe、Ad-HBpol、Ad-HBx and control adenovirus Ad-GFP. Realtime R-PCR and westernblot was used to explore the relationship between seven of HBV proteins and Apogenes. The results showed that recombinant adenovirus were successfully constructed,which could effectively infect target cells and express corresponding proteins. Ascompared to Ad-GFP infected Huh7and HepG2cells, ApoAII, ApoF and ApoH wasdown-regulated by HBs, MHBs and HBpol, respectively, tested by real time RT-PCR,which was coincident wih HBV level. The protein level of ApoAII, ApoF and ApoHwere further detected by western blot, the results indicated that ApoAII wasdown-regulated by HBs, which was coincident wih transcriptional level. While ApoFand ApoH was up-regulated by MHBs and HBpol, the exact mechanism was unclear,we consider there are transcription, post-transcription, translation and post-translationlevels involved in these course.
In the third part of this study is to elucidate the mechanism of HBV, expeciallyHBx induced hepatic steatosis via NQO1repression. The amount of intracellular ROSlevel and superoxide anion were detected, the results indicated that HBx-mediatedNQO1expression could increase the generation of intracellular ROS and superoxide.The intracellular GSH levels were measured and indicated that high levels of ROScould exhaust cellular antioxidant enzymes. To investigate whether conditions ofoxidative stress induced by HBx could predispose the cells to apoptosis,Annexin-V/PI dual staining for flow cytometric analysis was used to measure an earlyevent of apoptosis. The results showed that HBx expression increased apoptoticsusceptibility of the cells. To address directly whether HBx impairs cellular defensesagainst exogenous pro-oxidant stimuli, cells were exposed to increasingconcentrations of H2O2and cell viability measured. It caused much more cell death inHBx-expressing Ad-HBx cells than the control Ad-GFP cells. In addition,HBx-induced Δψm loss and ATP depletion is reversible by NQO1over-expression or DAC treatment.
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。
Infection of human hepatitis B virus (HBV) resulted in acute and chronichepatitis, and closely related to cirrhosis and hepatocellular carcinoma (HCC).Recently, with the increasing prevalence of Nonalcoholic fatty liver disease (NAFLD),hepatic steatosis along with chronic viral hepatitis B in persons become a commonphenomenon. Therefore, studies on the interaction between hepatic steatosis and HBVbecome more and more important. However, the exact association of HBV infectionand hepatic steatosis remains elusive. Previous studies from our laboratory haveshown that the protein level of Apolipoprotein is marked affected and NQO1issignificantly lower by using a proteomic approach and MALDI-TOF/TOF MSanalysis. These proteins may play important roles in lipid metabolism and progressionof hepatic steatosis. And in that basis, we investigated the effect of HBV protein(LHBs、MHBs、HBs、HBc、HBe、HBpol、HBx) on Apolipoprotein (Apo) expression,and further investigated the mechanism of HBx involvement in hepatic steatosis viaNQO1repression. These data may suggested a mechanism for HBV-associatedpathogenesis and provide theoretical basis for the effective prevention and treatmentof fatty liver.
The first part of this study is to investigate whether HBV affected transcriptionApo genes. HBsAg and HBeAg were measured by ELISA kit to confirmed HBVreplication and expression on stable HBV-producing hepatoma cell line and transienttransfection cells. Realtime RT-PCR to examine the16Apo genes transcriptional levelof HepG2.2.15and HepG2cells. And further confirmed by transient transfectionassay. The results showed that the transcriptional level of ApoAI，ApoAII，ApoAV，ApoB，ApoCIII，ApoE，ApoF，ApoH，ApoJ，ApoL1and ApoM is marked lowerin HepG2.2.15cells, while ApoD is significantly higher compared with that in control cell line. Besides, the effect of HBV on Apo genes transcriptional level istime-dependent.
In the second part of this study, AdEasy XL System was used to constructrecombinant adenovirus Ad-LHBs、Ad-MHBs、Ad-HBs、Ad-HBc、Ad-HBe、Ad-HBpol、Ad-HBx and control adenovirus Ad-GFP. Realtime R-PCR and westernblot was used to explore the relationship between seven of HBV proteins and Apogenes. The results showed that recombinant adenovirus were successfully constructed,which could effectively infect target cells and express corresponding proteins. Ascompared to Ad-GFP infected Huh7and HepG2cells, ApoAII, ApoF and ApoH wasdown-regulated by HBs, MHBs and HBpol, respectively, tested by real time RT-PCR,which was coincident wih HBV level. The protein level of ApoAII, ApoF and ApoHwere further detected by western blot, the results indicated that ApoAII wasdown-regulated by HBs, which was coincident wih transcriptional level. While ApoFand ApoH was up-regulated by MHBs and HBpol, the exact mechanism was unclear,we consider there are transcription, post-transcription, translation and post-translationlevels involved in these course.
In the third part of this study is to elucidate the mechanism of HBV, expeciallyHBx induced hepatic steatosis via NQO1repression. The amount of intracellular ROSlevel and superoxide anion were detected, the results indicated that HBx-mediatedNQO1expression could increase the generation of intracellular ROS and superoxide.The intracellular GSH levels were measured and indicated that high levels of ROScould exhaust cellular antioxidant enzymes. To investigate whether conditions ofoxidative stress induced by HBx could predispose the cells to apoptosis,Annexin-V/PI dual staining for flow cytometric analysis was used to measure an earlyevent of apoptosis. The results showed that HBx expression increased apoptoticsusceptibility of the cells. To address directly whether HBx impairs cellular defensesagainst exogenous pro-oxidant stimuli, cells were exposed to increasingconcentrations of H2O2and cell viability measured. It caused much more cell death inHBx-expressing Ad-HBx cells than the control Ad-GFP cells. In addition,HBx-induced Δψm loss and ATP depletion is reversible by NQO1over-expression or DAC treatment.
引文
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