CD81介导滋养细胞抑制内皮细胞eNOS参与子痫前期血管内皮功能障碍的机制研究

Trophoblast shallow invasion and maternal vascular endothelial cell spasm are considered as the pathophysiological changes of preeclampsia (PE). Recently, studies have focused on investigating the molecules that are not only contributed to extravillous trophoblast (EVT) shallow invasion and impaired placentation, but also are lead to maternal vascular endothelial cell dysfunction in the second or third trimester. Our previous study found that tetraspanin CD81 is involved in the development of early onset PE: CD81 is significantly upregulated in EVT, syncytiotrophoblasts, the cells in villous core and peripheral sera of patients with PE; Also, CD81 inhibits trophoblast invasion and causes endothelial cell activation and interrupted angiogenesis. In addition, overexpression of CD81 downregulates p-Akt and eNOS expression in trophoblasts. We suppose that the abnormally high expressed CD81 in trophoblasts acts on endothelial cells via microparticles or exosomes to inhibit PI3K/Akt signaling pathway and eNOS expression, then effects vasorelaxation and endothelial cells permeability that are related to PE development. In this project, molecular information transmission between cells and in vivo animal experiments are studied to clarify the trophoblastic CD81 leads to maternal endothelial cell dysfunction through the PI3K/Akt/eNOS mechanism, providing the new targets for prevention and treatment of PE.

子痫前期（Preeclampsia, PE）病因不明，胎盘浅着床和母体血管内皮损伤是PE公认的两个病理生理变化。近年的研究致力于寻找孕早期使绒毛外滋养细胞（EVT）侵蚀不足致胎盘浅着床的关键分子及其引起母体孕中晚期血管内皮损伤的因素。我们最近的研究发现CD81参与早发型PE发病：它在患者EVT、合体滋养细胞及绒毛间质细胞高表达，而且在外周血清中也升高，还表明CD81抑制EVT侵蚀，也引起内皮细胞激活和成环障碍。另还发现CD81降低滋养细胞中eNOS和p-Akt的表达。我们假设：CD81抑制EVT侵蚀导致胎盘浅着床，孕中晚期由绒毛细胞碎片或外泌体释放到血清中的CD81增加，使内皮细胞PI3K/Akt通路抑制，从而降低eNOS表达，导致内皮细胞功能障碍。本研究将从细胞间分子信息传递和动物实验入手，阐释滋养细胞通过CD81/PI3K/Akt/eNOS导致血管内皮功能障碍，为PE防治提供新靶点。

子痫前期（Preeclampsia, PE）是妊娠期常见的特有的并发症且目前病因不明。PE发病的“二阶段”理论认为：PE发病的第一阶段为胎盘发育异常，第二阶段为母体全身血管内皮细胞功能受损，引发高血压、尿蛋白及多器官功能紊乱等临床表现。我们前期研究发现，CD81在重度PE（sPE）患者的胎盘和外周血清中均高表达，且CD81抑制滋养细胞的侵袭并引起内皮细胞功能紊乱。本项目研究中，我们首先明确CD81在sPE患者的胎盘中和血清中高表达，同时eNOS和p-Akt S473在sPE患者胎盘中表达均下降。其次，发现高表达CD81的滋养细胞上清引起内皮细胞激活，表现为ICAM-1和VCAM-1表达增加，同时使原代HUVECs细胞的成环能力下降、细胞凋亡增加、细胞侵袭能力下降和细胞的通透性增强。进一步的分子机制探究发现，高表达CD81的滋养细胞上清抑制HUVECs中eNOS的表达，反之干扰CD81小片段（siCD81）抑制CD81表达的滋养细胞上清能够促进eNOS的表达。信号通路探究发现，高表达CD81的滋养细胞上清抑制HUVECs中PDK1和p-Akt S473表达，反之siCD81则作用相反。利用PI3K/Akt通路抑制剂LY294002预处理HUVECs，将siCD81-media作用于HUVECs，发现内皮细胞中eNOS和Akt S473的表达均逆转下降，提示CD81通过PI3K/PDK1/Akt信号通路来抑制内皮细胞中eNOS的表达。最后，通过动物实验表明，高表达CD81腺病毒（Ad-CD81）尾静脉注射大鼠和胎盘特异性高表达CD81的转基因小鼠均表现出PE样的表型，表现为高血压、尿蛋白、胎儿吸收增加和子宫螺旋动脉重塑障碍。另外，Ad-CD81模型大鼠的胎盘中eNOS和p-Akt S473表达均显著下调。综上所述，滋养细胞高表达CD81通过PI3K/PDK1/Akt信号抑制内皮细胞中的eNOS表达，从而引起内皮细胞功能异常，参与PE的发病。