乙型肝炎病毒X蛋白与COXIII结合导致肝细胞炎症损伤机制的研究

HBV X protein(HBx), a viral protein encoded by HBV, is essential for HBV replication and acts as a key factor in the development of inflammatory injury and hepatocarcinoma induced by HBV. However, the mechanism and details of HBx on inflammatory injury in human hepatocytes, which may also be a main reason for oncogenesis of hepatocarcinoma, remain unclear. Our previous study had reported the finding of a new HBx-interacting protein,cytochrome c oxidase subunit III(COXⅢ) which is important for mitochondrial function, by yeast two hybrid system. This achievement builds the basement of follow-up researches for HBx pathogenetic mechanism. In this study, a confocal microscopy will be used to observe the functional location of HBx interacting with COXIII in innermembrane of mitochondria and the ultrastructual changes caused by interaction between HBx and COXIII. A complex mixed by cytochrome c oxidase and liposome will mimic cytochrome c oxidase in mitochondrial membrane in hepatocytes,so the proton pump function of COXIII can be studied in a relatively independent way.The purified HBx will be added to the complex to inhibit proton pump function of COXIII. Cytochrome c oxidase function interferred by HBx will also be examined in both the complex and hepatocytes. The open of mitochondria permeability transition pore(MPTP) will be measured by detection of calcium level in human hepatocytes with or without HBx expression.The function injury of mitochondria will be examined as a comprehesive one, distinguished from respectively independent function of COXIII, cytochrome c oxidase and MPTP. Inflammatory changes in both human hepatocytes and mouse liver will be assessed as final evaluations for injury caused by HBx. A HBx siRNA and Cyclosporin A which is a specific inhibitor for MPTP will be applied to reverse the inflammatory injury induced by HBx. This study is designed to give an interpretation for the mechanism of inflammatory injury induced by HBx in human hepatocytes，and will make HBx a target for treatment of patients with HBV infection.

乙型肝炎病毒致肝脏慢性炎症和细胞癌变的具体机制目前仍未明确，乙肝病毒X蛋白（HBx）在此过程中发挥关键作用。课题组前期研究首次证实了细胞色素C氧化酶亚单位III（COXIII）是HBx的体内结合蛋白，COXIII在维持线粒体正常功能中发挥重要作用。本课题将在共聚焦显微镜下观察HBx同COXIII结合后线粒体超微结构的改变，检测HBx对COXIII质子传递功能及细胞色素C氧化酶功能的影响，观测由此诱发的线粒体通透性转换孔（MPTP）开放造成细胞内钙超载所带来线粒体功能的级联损害，评估人肝细胞和小鼠肝脏组织在HBx作用下炎症损伤指标。使用HBx siRNA和MPTP特异性抑制剂-环孢素A阻断HBx的作用，再次评估以上指标的改变。本课题将有助于深入阐明HBx导致肝细胞炎症损伤的过程和机制，为针对HBx的相关治疗开创新思路。

乙型肝炎病毒致肝脏慢性炎症和细胞癌变的具体机制目前仍未明确，乙肝病毒X蛋白（HBx）在此过程中发挥关键作用。课题组前期研究首次证实了细胞色素C氧化酶亚单位III（COXIII）是HBx的体内结合蛋白，COXIII在维持线粒体正常功能中发挥重要作用。本课题在共聚焦显微镜下观察HBx同COXIII结合后线粒体超微结构的改变；检测HBx对细胞色素C氧化酶功能的影响，观测由此诱发的线粒体通透性转换孔（MPTP）开放造成细胞内钙超载所带来线粒体功能的级联损害，评估细胞在HBx作用下炎症损伤指标；使用MPTP特异性抑制剂——环孢素A阻断HBx的作用，再次评估以上指标的改变；并进一步使用酵母双杂交及免疫共沉淀技术筛选HBx同COXIII的结合位点。研究结果显示：1、利用慢病毒载体成功构建稳定表达HBx的人肝细胞株HL7702/HBx与肝癌细胞株HepG2/HBx；利用脂质体成功构建瞬时表达HBx的人肝细胞株 HL7702/pcDNA3.1-HBx。 2、在HL7702/HBx与HepG2/HBx细胞中，HBx与COXIII共定位，引起细胞线粒体嵴稍肿胀，细胞形态及结构无明显改变。3、稳定表达HBx的HL-7702细胞与肝癌细胞HepG2中COXIII蛋白的表达增加，COX酶活性增高，而在瞬时转染HBx的HL-7702细胞中COX酶活性下降。4、HBx致HL7702/pcDNA3.1-HBx细胞ATP水平及线粒体膜电位下降，并通过调节MPTP从而引起肝细胞胞浆钙离子超载。5、HBx通过调节MPTP增加了HL-7702与HepG2细胞内的ROS水平，一方面使得HL7702/HBx与HepG2/HBx细胞表达炎症介质环氧化酶-2（COX-2）增加，上调HL7702/pcDNA3.1-HBx细胞NF-κBP65、RIP3、TNFα的表达水平，促进细胞的增殖；另一方面增加了HL7702/pcDNA3.1-HBx细胞对氧化应激的敏感性，而在氧化应激的刺激下，HBx蛋白通过调节MPTP使Bax蛋白向线粒体转位，促进线粒体细胞色素C的释放，引起细胞的凋亡。6、HBx蛋白同COXIII特异性结合，其结合位点位于HBx蛋白的72~117位氨基酸，以上结果提示： HBx蛋白能够与COXIII共定位于线粒体，影响COX酶活性，使线粒体功能改变， MPTP开放，导致肝细胞胞浆钙离子超载、ROS表达增加，进