【作者】 殷一旋；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2010， 硕士

【摘要】 乙型肝炎严重威胁着人类的健康,据统计全世界的乙肝病毒携带者有3.5亿之众。而乙肝病毒携带者一旦发展为慢性乙肝,就很可能恶变为肝硬化和肝细胞性肝癌。在慢性乙型肝炎进展为肝硬化、肝细胞癌的进程中,男性患者进展的速度比女性快,流行病学调查结果显示男性及绝经后女性更易患慢性乙肝相关性肝硬化及肝癌。因此我们认为在感染了乙肝病毒后,男、女性慢性乙肝患者在向肝硬化及肝癌演变过程中有着本质的区别,而对这种疾病进展的性别差异目前还没有一致的解释机制。为探索不同性别中慢性乙型肝炎疾病进展差异的机制,我们采用双向荧光差异凝胶电泳(two-dimensional difference gel electrophoresis,2-D DIGE)的方法分别比较雌、雄HBV转基因鼠与同性别C57BL／6小鼠的肝脏组织中蛋白质表达的差异,结果显示载脂蛋白A-I (Apo A-I, apolipoprotein A-I)总表达量在雌、雄HBV转基因鼠肝组织中均下降。由于血循环中Apo A-I的水平与慢性乙肝及其相关原发性肝细胞癌相关,低水平的Apo A-I提示有严重的肝损伤,因此我们发现雌雄HBV转基因鼠肝中Apo A-I表达下调,可能是因为HBV在体内抗原表达引起肝细胞损伤并扰乱肝细胞脂质代谢。更有趣的是,我们使用2-D western blotting方法研究HBV转基因鼠肝组织及慢性乙肝病人血清中Apo A-I的表达,发现Apo A-I的亚型3表达量在雄性HBV转基因鼠肝组织及男性慢性乙肝病人血清中均有明显增加,而雌性转基因鼠肝组织及女性病人血清中Apo A-I的各亚型比例均无明显改变。Apo A-I主要在肝脏中合成并被认为存在翻译后修饰。雄性HBV转基因鼠肝脏及男性慢性乙肝病人血清中Apo A-I各亚型表达量比例的改变反映了不同的亚型有其不同的翻译后修饰,因此他们的功能和特征也不同。近来研究发现在慢性乙肝相关性肝癌患者及肝癌转基因鼠模型的血清中存在高浓度的氧化型Apo A-I。我们进一步的实验结果显示在雄性转基因鼠肝组织及男性病人血清中增加的Apo A-I的亚型3为氧化修饰型Apo A-I,而女性却没有,可能是因为女性体内的雌激素及其衍生物的作用使Apo A-I的氧化过程被阻断或减弱。因此雄性HBV转基因鼠肝脏及慢性乙肝病人血清中Apo A-I亚型3的升高可能为男性乙型肝炎进展较女性更快的现象提供新的思路。以上这些结果提示慢性乙肝病毒感染扰乱了Apo A-I亚型比例,进而阻碍了Apo A-I整体功能的发挥。因此,我们认为这种选择性氧化修饰的Apo A-I亚型可作为病理标志用于进一步研究慢性乙肝的发病机制；而在临床上,Apo A-I亚型的特异性抗体结合其他传统标志物可用于慢性乙肝的预后评估。更多还原

【Abstract】 Chronic hepatitis B (CHB) is a major risk factor for hepatocellular carcinoma (HCC). One interesting feature of CHB-related HCC is the male predominance, with a male-to-female ratio of 5-7:1. There is similar or even more pronounced gender disparity in hepatitis B virus (HBV) transgenic mouse models. All these facts indicate that HBV influences the function of male and female host cells in different ways. The mechanisms that account for this gender disparity remain unclear. We need some new points of view and methods to study this problem.In this study, we focused on 6-8-week-old male and female HBV-transgenic (HBV-Tg) mouse liver, which represents the typical age at which early pathological lesions are detectable. Because of the great sensitivity and dynamic range that are afforded by fluorescent tags Cy2, Cy3 and Cy5, two-dimensional difference gel electrophoresis (2-D DIGE) can give much greater accuracy of quantization. To obtain more insight into the underlying mechanisms of the gender disparity of CHB progression,2-D DIGE was employed to compare the liver proteome of C57BL/6 and HBV-Tg mice in males and females.We identified 8 differently expressed proteins in male HBV-Tg mice and 12 in female HBV-Tg mice. Apolipoprotein A-I (Apo A-I) was found to be down-regulated in male and female HBV-Tg mouse liver., which indicated that HBV antigen expression in vivo might cause liver cell injury and disturb liver cell lipid metabolism.HBV can affect liver cell lipid metabolism in male and female HBV-Tg mice, but there are some interesting differences between the two sexes. Our results indicated that HBV antigen expression-mediated Apo A-I alteration occurred not only in its total protein expression level but also in the pattern of Apo A-I isoform expression in male HBV-Tg mouse liver, whereas we did not find any changes in the pattern of Apo A-I isoform expression in female HBV-Tg mouse liver. Furthermore, we got accordant results in male and female CHB patient sera, but did not in CHC patient sera. Apo A-I is initially synthesized as a preproprotein and can be modificated at the post-translation level。The heterogeneous alteration of the pattern of Apo A-I isoform expression in male HBV-Tg mouse liver reflects different post-translational regulation of different isoforms of Apo A-I, which may be correlated with specific features or functions of the isoforms.Finally, we found that the oxidized Apo A-I was increased in male HBV-Tg mouse liver and the increase in the Apo A-I isoform 3 in liver of male HBV-Tg mice may represent a highly oxidized Apo A-I isoform. This process of over-oxidizing Apo A-I may be blocked or deduced by estradiol or its derivatives in female HBV-Tg mouse liver. The different pattern of Apo A-I isoforms observed in male and female transgenic mouse liver may be the result of an interaction between oxidative stress induced by HBV antigen expression and sex hormones in vivo. The increase in Apo A-I isoform 3 in male HBV-Tg mouse liver and CHB patient serum may provide a new clue to explain the male gender susceptibility to CHB progression.Although it is not clear at present whether the occurrence of these modifications has a causal role or simply reflects secondary epiphenomena, the selectively modified Apo A-I isoforms may be considered to be a pathological hallmark that could extend our knowledge of the molecular pathogenesis of CHB. Development of antibodies that specifically recognize the isoforms of Apo A-I may prove to be useful, in combination with other traditional markers, as a more efficient way to evaluate the prognosis of CHB.更多还原