IL-6对NTCP介导HBV胎盘细胞感染及细胞间直接感染的调节

Mother-to-infant transmission is still reported as the bottleneck for further reducing HBV infection in China. As crucial barrier between mother and fetus, placenta infected with HBV has been considered as one important route of HBV intrauterine transmission. NTCP has been proved to be an essential receptor for HBV infection; additionally IL-6 can influence HBV infection via inhibiting NTCP expression. During our previous study, expression of NTCP and IL-6 in placenta has been confirmed; an association of serum IL-6 level with HBV intrauterine infection has been displayed. We speculated that NTCP mediate HBV infection in placenta and cell-cell contact transfer and IL-6 regulate HBV intrauterine transmission via influencing NTCP expression and membrane translocation. To clarify those hypotheses, the following works will be carried out in this program, 1) to confirm the NTCP function in HBV infection and intrauterine transmission on three levels of placenta tissue, primary cells and singlenueleotide polymorphism; 2) exploiting fluorescent living cell tracker (CMFDA/PKH26) and the membrane-permeant fluorogenic biarsenicals FlAsH/ReAsH-EDT2 labeling system, to observe the cell-cell contact transfer of HBV and obtain 3D image; 3) with expression plasmid of NTCP with two-terminal labels and confocal microscopy, to investigate regulation of IL-6 on expression and membrane translocation of NTCP, as well as influence on HBV infection. With current program, we will identify the underlying function and regulatory mechanism of NTCP on HBV infection in placenta, which will expand our views about intrauterine transmission, help looking for influencing factors and provide potential intervention model targeting NTCP.

母婴传播是我国进一步降低乙肝感染率的关键。胎盘感染是宫内传播的最重要途径。NTCP作为HBV感染的受体已逐步得到共识，IL-6可影响NTCP介导的HBV感染。我们先前证明胎盘表达NTCP及IL-6，血清IL-6水平与HBV宫内感染具有相关性，据此假设NTCP可介导HBV胎盘感染及细胞间感染，IL-6通过调节NTCP表达、膜转位而影响乙肝宫内传播。本课题拟①在组织、细胞及基因SNP水平证明NTCP在HBV感染胎盘中的作用；②采用活细胞标记及TC-FlAsH/ReAsH染色标记系统观察NTCP介导的HBV细胞间感染；③探讨IL-6对胎盘NTCP表达的调节，采用NTCP膜内外双标记表达质粒检测IL-6/PKC对NTCP膜转位、受体功能的调节。藉此初步阐明NTCP在HBV感染胎盘、宫内传播中作用及调节机制，丰富乙肝宫内传播的理论，寻找可能的影响因素，为制定新的以NTCP为靶点的干预模式提供依据。

无论从乙型肝炎病毒（HBV）感染的流行病学还是病毒基因进化树分析均证明，母婴传播是我国HBV流行的重要原因之一。HBV母婴传播还是不良家族聚集性感染的主要原因。感染后病毒清除非常困难且肝硬化和肝癌的发生是普通人群的100倍以上。实现乙肝母婴“零”传播是达到WHO消除肝炎目标的关键所在，高病毒载量孕妇孕期抗病毒干预可显著降低母婴传播风险，但高病毒载量孕妇发生宫内感染的机制仍不清楚。本课题以HBV宫内传播为背景，探讨HBV经胎盘发生宫内传播的机制，丰富HBV母婴传播的机制，为研究其他病毒宫内传播机制提供参考。.本项目研究发现：①在发生母婴传播的慢性HBV感染母婴对，HBV感染不良结局家系中，NTCP rs2296651位点 GG的频率与感染呈负相关，且与不良结局家族中肝癌的发生密切相关；②HBV病毒通过上调NTCP表达，促进自身感染及复制；③IL-6调节NTCP表达，参与HBV感染滋养细胞过程中细胞自噬、炎症的调节；④HBV-x基因参与滋养细胞中NTCP上调、影响滋养细胞自噬及迁移功能，促进宫内传播的发生。本课题结果发现HBV病毒通过上调受体、利用细胞自噬来促进病毒感染滋养细胞及复制，通过影响滋养细胞炎症、迁移促进感染传播，丰富了HBV宫内传播的机制。且这些作用均与病毒载量正相关，也为孕期抗病毒干预提供分子基础依据。.综上，在本期国科金的支持下，课题组围绕HBV宫内感染的机制，以经胎盘感染为核心，探索HBV宫内感染的分子机制，丰富HBV宫内传播的机制，为研究其他病毒宫内感染机制研究提供参考。