【摘要】：乙型肝炎病毒（Hepatitis B virus，HBV）導(dǎo)致急慢性乙型肝炎，并與肝硬化和肝細(xì)胞癌（Hepatocelluar carcinoma，HCC）的發(fā)生發(fā)展密切相關(guān)。近年來由于脂肪性肝病（Fatty liver disease，F(xiàn)LD）的發(fā)病率逐年增加，導(dǎo)致脂肪肝與慢性乙型肝炎（chronic hepatitis B，CHB）合并存在的病例明顯增多，對兩者相關(guān)性的研究逐漸受到重視。但是迄今為止，HBV與脂肪肝的關(guān)系尚未完全闡明。本課題組先前通過蛋白質(zhì)組學(xué)和MALDI-TOF/TOF MS技術(shù)研究發(fā)現(xiàn)HBV影響肝細(xì)胞載脂蛋白（Apolipoprotein，Apo）的表達(dá)，并降低NAD(P)H醌氧化還原酶1（NAD(P)H dehydrogenase:quinone1，NQO1）的表達(dá)，現(xiàn)有研究表明上述因子參與脂肪酸的合成和代謝，對肝臟脂質(zhì)代謝動態(tài)循環(huán)平衡的維持起著重要的作用。本研究在此基礎(chǔ)上，探討HBV編碼蛋白（LHBs、MHBs、HBs、HBc、HBe、HBpol、HBx）與特定宿主細(xì)胞中載脂蛋白表達(dá)的對應(yīng)關(guān)系，進(jìn)一步探討HBV影響NQO1致肝細(xì)胞脂肪化的機(jī)制，有助于更深入了解HBV的致病機(jī)制，為有效地進(jìn)行脂肪肝的防治提供理論基礎(chǔ)。 本研究第一部分旨在探討HBV整體水平對Apo基因的影響。通過對HBV細(xì)胞株和pRep-HBV質(zhì)粒瞬時轉(zhuǎn)染細(xì)胞中HBsAg及HBeAg的檢測，，確定HBV基因組的正常復(fù)制和表達(dá)；通過realtime RT-PCR的方法，檢測了16種Apo基因在HepG2.2.15及HepG2細(xì)胞中表達(dá)的差異，并進(jìn)一步用瞬時轉(zhuǎn)染的方法在Huh7、HepG2和Hep3B細(xì)胞進(jìn)行驗證，結(jié)果表明ApoAI，ApoAII，ApoAV，ApoB，ApoCIII，ApoE，ApoF，ApoH，ApoJ，ApoL1和ApoM和對照組相比轉(zhuǎn)錄水平均明顯降低，APOD的轉(zhuǎn)錄水平和對照組相比明顯升高；通過檢測不同時間點(diǎn)HBV表達(dá)量對上述Apo基因轉(zhuǎn)錄水平的影響，證實(shí)HBV表達(dá)量對Apo基因的表達(dá)影響具有時間效應(yīng)。 本研究第二部分應(yīng)用AdEasy XL System構(gòu)建HBV七種編碼蛋白的重組腺病毒表達(dá)載體Ad-LHBs、Ad-MHBs、Ad-HBs、Ad-HBc、Ad-HBe、Ad-HBpol、Ad-HBx及對照空病毒Ad-GFP，探討HBV編碼蛋白對特定宿主細(xì)胞中Apo基因表達(dá)的對應(yīng)關(guān)系。通過對腺病毒包裝過程的監(jiān)測和Westernblot檢測，成功構(gòu)建可分別表達(dá)HBV LS、MS、S、Core、E、Pol、 X蛋白的重組腺病毒；通過realtime RT-PCR檢測HBV七種編碼蛋白對Apo基因的表達(dá)影響，我們發(fā)現(xiàn)HBs下調(diào)ApoAII的轉(zhuǎn)錄水平，MHBs下調(diào)ApoF的轉(zhuǎn)錄水平，HBpol下調(diào)ApoH的轉(zhuǎn)錄水平，且均與HBV整體水平結(jié)果一致；通過western blot進(jìn)一步檢測ApoAII、ApoF和ApoH基因蛋白水平的變化，發(fā)現(xiàn)HBs下調(diào)ApoAII蛋白水平，與轉(zhuǎn)錄水平一致；MHBs和HBpol分別上調(diào)ApoF和ApoH的蛋白水平，與轉(zhuǎn)錄水平不一致，我們考慮可能存在轉(zhuǎn)錄、轉(zhuǎn)錄后水平、翻譯和翻譯后水平的多種組合調(diào)控，具體機(jī)制有待進(jìn)一步的研究。 本研究第三部分旨在探討HBV特別是HBx抑制NQO1致肝細(xì)胞脂肪化的機(jī)制。通過對活細(xì)胞內(nèi)源性ROS和超氧化物的檢測，證實(shí)HBx通過抑制NQO1的表達(dá)從而誘發(fā)細(xì)胞的氧化應(yīng)激；通過對細(xì)胞內(nèi)抗氧化酶還原性谷胱甘肽GSH的檢測，表明過量的ROS產(chǎn)生可能會破壞細(xì)胞內(nèi)的抗氧化防御體系；應(yīng)用Annexin-V/PI雙染法檢測細(xì)胞凋亡的發(fā)生，證實(shí)過量的ROS蓄積引起細(xì)胞凋亡；同時通過采用CCK-8檢測H2O2處理后細(xì)胞的生存率，表明HBx抑制NQO1表達(dá)使細(xì)胞對H2O2引起的細(xì)胞毒性反應(yīng)更敏感；通過線粒體Δψm和ATP水平的檢測我們發(fā)現(xiàn)NQO1參與了HBx誘發(fā)的線粒體損傷作用。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R512.62;R575

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳從新;劉波;楊家宏;劉克萬;郭順明;周天仇;;拉米夫定治療失代償期肝硬化伴乙型肝炎相關(guān)性腎炎成功3例報道[J];實(shí)用肝臟病雜志;2007年04期

2 夏國豪,張新年,林永剛;乙肝病毒感染與惡性淋巴瘤的化療[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊);1998年01期

3 劉奇才;郜峰;程祖建;廖冬妹;歐啟水;;戊型肝炎病毒感染與胰腺癌發(fā)生的關(guān)系[J];檢驗醫(yī)學(xué)與臨床;2007年11期

4 于曉輝,趙連三,吳雄志,馬穎,許倩;乙型肝炎合并急性胰腺炎六例臨床分析[J];臨床內(nèi)科雜志;2004年10期

5 姚佳,張祖輝,劉秉文;載脂蛋白CⅢ研究進(jìn)展[J];現(xiàn)代預(yù)防醫(yī)學(xué);2002年01期

6 金洲祥,黃生福,張威;重癥肝炎并發(fā)急性胰腺炎25例[J];世界華人消化雜志;2004年08期

7 杜會芹;尹苗;葉紅燕;商允菊;戴學(xué)棟;荊文;張晾;肖寧;李繼峰;潘杰;;apoE/LDLR雙基因突變小鼠肝臟脂代謝相關(guān)基因的表達(dá)研究[J];中華病理學(xué)雜志;2007年11期

8 趙連三,劉曉松,張智翔,王錦蓉,劉麗,雷秉鈞;乙型肝炎病毒感染經(jīng)精子傳播的可能性研究[J];中華傳染病雜志;1998年03期

9 董菁,成軍,王勤環(huán),王剛,施雙雙,劉妍,夏小兵,斯崇文;外周血中乙型肝炎病毒截短型囊膜蛋白基因的克隆化與序列分析[J];中華肝臟病雜志;2001年03期

10 鄧子德，庚慧鳴，何達(dá)秋，姚集魯;脂肪肝患者中的HBV／HCV感染及其臨床意義[J];中華肝臟病雜志;1996年03期

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