细胞与线粒体自噬和滋养细胞损伤及其与HBV感染宫内传播的关系研究

Chronic hepatitis B infection, caused by mother-to-infant transmission is difficult to cure, and the morbidity of liver cirrhosis and hepatocarcinoma is over 100 times higher than the normal population. With nucleotide analogue intervening during pregnancy and combined prophylaxis of HB vaccine and HBIG to newborns of chronic HBV infected (CHB) mothers, the vertical transmission has been decreased in a great degree. However, there is still about 2%-3% infants obtaining chronic infection owing to intrauterine transmission, which is the bottleneck to eradicate the infection of HBV. As the first line of placenta, trophoblast is the critical defense barrier of HBV transmitting to fetus. In our previous investigation, damage in placenta basement membrane and mitochondrial swelling had been observed in placenta of CHB mothers. Furthermore, autophagy change and upregulation of NTCP expression in trophocytes infected with HBV has been displayed, as well as oxidative stress, which suggest the association of HBV infection, autophagy and mitochondrial damage influence trophocytes barrier. According to our previous clinical work and molecular study, in this proposal, the impact of the typical autophagy signaling pathway or NTCP-APOA1-HBx-Beclin1 pathway on autophage and virus replication will be clarified; in addition, the influence of interaction between HBV and autophage on mitochondria will be illuminated, as well as barrier function of trophocytes. With this study, the theory of HBV mother-to-infant transmission can be expanded and possible intervention method can be put forward.

母婴传播形成的HBV感染难于清除且肝硬化和肝癌发生率是普通人群100倍以上。联合免疫预防和孕期核苷类似物干预后仍有2%~3%发生宫内感染，是实现母婴零传播的壁垒。乙肝病毒突破滋养细胞屏障是宫内感染关键。我们前期发现感染者胎盘滋养细胞基底膜及线粒体损伤，滋养细胞系实验证实HBV可影响细胞自噬，上调细胞膜NTCP表达，并诱导线粒体发生氧化应激，提示病毒感染和滋养细胞自噬及线粒体自噬相互关联，影响滋养细胞屏障功能。基于我们前期临床实践和上述发现，拟进一步从分子、细胞模型、胎盘组织及动物模型不同水平阐明HBV通过经典自噬途径或NTCP介导APOA1-HBx-Beclin-1途径影响细胞自噬，促进HBV病毒复制；揭示病毒感染与自噬相互作用造成的线粒体损伤、线粒体自噬，最终改变滋养细胞屏障功能、发生宫内传播的机制。通过该课题的完成将丰富乙肝母婴传播理论、提供可能的干预方向。

母婴传播是我国HBV流行的重要原因之一。幼年时期的HBV感染 85%以上不易被机体清除，慢性化率、肝硬化及肝癌发生率远高于普通人群。HBsAg阳性母亲的新生儿产后联合免疫可有效阻断分娩过程和产后的感染，但仍有超过10%的婴儿因宫内感染导致慢性乙型肝炎病毒感染。基于我国庞大的人口基数、相对高的育龄期妇女HBV感染率、母婴传播仍是我国最终消除HBV感染的瓶颈。阻断宫内感染更是进一步降低我国乙肝流行、减少乙肝相关终末期疾病发生的关键所在，但是宫内感染发生的机制仍不清楚。. 本项目的研究结果显示：高病毒载量乙肝产妇胎盘组织高表达炎症指标NLRP3和IL-1β，滋养细胞内存在乙肝相关蛋白的表达和线粒体损伤，而孕期行抗病毒治疗的乙肝产妇胎盘组织炎症和线粒体损伤状态得到改善。体外实验HBV与滋养细胞系共培养模型中发现，高载量HBV可抑制细胞线粒体自噬，降低线粒体膜电位，升高细胞内ROS水平；且HBV感染受体NTCP的表达也随着HBV病毒载量的增加而增加。蛋白组学和代谢组学结果也表明高病毒在载量乙肝产妇胎盘组织与正常产妇胎盘组织的差异蛋白或者代谢物主要富集在线粒体相关通路上。. 综上，本课题从分子水平证明了高病毒载量乙肝孕妇孕期抗病毒干预的必要性；明确了线粒体损伤在HBV宫内感染中的重要作用，揭示了宫内感染可能的分子机制，为其他病毒的宫内感染提供了研究线索，为寻求新的阻断措施提供了理论依据和方向。