S100A-Anx复合体及RE-出胞复合体介导滋养细胞内HBV出胞的分子机制

Transcytosis through placental trophoblasts barrier has been confirmed as a crucial step for hepatitis B virus (HBV) to cause intrauterine transmission. The recent reports showed that the S100A-Anx complex mediates exocytosis of human papilloma virus (HPV) from human epithelial cells while the HBV in hepatocytes is located in intracellular multivesicular body (HBV-MVB) and secretes through recycling endosome (RE) route for exocytosis. Our previous study found that the expression levels of S100A8/A9/A12 proteins in both the placental tissues and neonatal sera of HBV carrying pregnant women with intrauterine transmission were significantly increased as well as the HBV copied in trophoblasts and secreated from the cells. However, the mechanism of HBV exocytosis from trophoblasts remains unknown. In this project, we will use HBV-infected Swan71 trophoblasts model to confirm the formation of HBV-MVB in trophoblasts during infection as well as recruit different S100A and Anx proteins to form S100A-Anx complex, and Rab11 and Sec/Exo proteins to form exocyst complex, by Rab5 or Rab7 on the surface of the HBV-MVB, which are subsequently combined with VAMP2/SNAP25/SYN proteins to form membrane fusion complex. All the research works will elucidate the molecular mechanism of HBV exocytosis from the infected trophoblasts through S100A-Anx complex-membrane fusion complex and RE-exocyst complex-membrane fusion complex mediated by PI3K/FAK signaling pathways and Ca2+-dependent cytoskeletal rearrangement, which have high creativeness and clinical significance.

突破胎盘滋养细胞屏障是乙肝病毒（HBV）宫内传播的关键。近年文献报道S100A-Anx复合体介导人上皮细胞中人乳头瘤病毒出胞，肝细胞内HBV位于多囊泡体（HBV-MVB）中并经循环内体（RE）途径出胞。我们前期研究发现HBV宫内感染孕妇胎盘及新生儿血清中S100A8/A9/A12蛋白表达显著升高、HBV可在滋养细胞中复制并外分泌，但出胞机制不明。本项目拟采用HBV感染Swan71滋养细胞模型，首先证明HBV感染细胞后形成HBV-MVB，通过HBV-MVB表面Rab5或Rab7招募不同S100A和Anx形成复合体、招募Rab11和Sec/Exo形成出胞复合体，然后结合VAMP2/SNAP25/SYN形成膜融合复合体，以阐明HBV经S100A-Anx复合体-膜融合复合体、RE-出胞复合体-膜融合复合体途径、PI3K/FAK通路介导Ca2+依赖细胞骨架重排出胞的分子机制，有较高创新性及医学意义。

HBV宫内传播是HBV母婴传播阻断失败及慢性HBV感染的主要原因。前期研究发现HBV可体内外感染穿越滋养细胞层导致母婴传播，蛋白质组学分析发现HBV宫内感染新生儿血清中S100A类蛋白表达较未发生宫内感染组明显升高、宫内感染组胎盘组织中S100A类蛋白表达也明显升高。本研究通过体内外实验（Swan71细胞系、原代滋养细胞、胎盘组织）构建了体外HBV感染穿越滋养细胞模型、研究了HBV感染滋养细胞出胞途径。发现HBV能感染穿越滋养细胞层，进入滋养细胞的HBV聚集于Rab7为标志的多囊泡体（multivesicular body，MVB）中形成HBV-MVB，共定位免疫荧光研究证实:滋养细胞内HbsAg与Rab7、S100A10、AnexinA2、VAMP2、SNAP25存在共表达，且HBV感染穿越滋养细胞层时细胞内Ca2+表达明显升高，而滋养细胞内HbsAg与Rab5、Rab11、S100A8、S100A9、S100A12、AnexinA1、AnexinA6无共表达；基因沉默滋养细胞中S100A10和Anexin2表达后，HBV穿越滋养细胞数量较对照组减少免疫组化结果显示发生HBV宫内传播胎儿胎盘组织中S100A8/ A9/A10/A12均表达升高，Western Blot结果证实HBV感染Swan71和原代滋养细胞中S100A8/A9/A10/A12也均表达升高。免疫细胞荧光染色证实：HBV感染滋养细胞中HbsAg与LC3和AnxA2存在共表达，透射电镜检查发现HBV感染Swan71滋养细胞中病毒颗粒及致密溶酶体均明显增多，且溶酶体中也包含较多的病毒颗粒。推测：滋养细胞内吞噬入胞的HBV大部分病毒被运送到溶酶体降解，只有少部分通过Rab7介导的S100A10-AnxsA2复合体- VAMP2/SNAP25/SYN膜融合复合体，激活膜钙通道介导Ca2+及MF/MT依赖细胞骨架重排被转运出胞。研究结果对阐明HBV宫内传播阻断措施研究提供理论依据。