mTORC2-AKT-IP3R介导的MAMs钙转运在妊娠肝内胆汁淤积症胎盘滋养细胞缺氧-复氧应激中的作用及机制研究

Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disorders during pregnancy. ICP is complicated with unpredictable fetal hypoxia and even intrauterine fetal death, which is extremely harmful to the fetus. The physiopathologic mechanism of fetal anoxia remains unclear. Our previous studies reported hypoxia-related dysregulation of mTOR in ICP placentas. mTORC2-AKT-IP3R could accurately regulate the Ca2+ transfer in Mitochondria Associated Membranes (MAMs), thus determine the stress intensity of endoplasmic reticulum and mitochondria in coping with hypoxia. This process plays a critical role in responding to hypoxia stress in most cells. Based on our previous research, this study will explore the biological function and mechanism of mTORC2, AKT and IP3R in regulating the Ca2+ transfer of MAMs by culturing the placental trophoblast cells in hypoxia-reoxygenation condition. Meanwhile, we will study the expression and activity of mTORC2-AKT-IP3R, as well as this signal pathway mediated MAMs Ca2+ transfer in ICP placentas. Our results will help to reveal the physiopathologic mechanism of intrauterine fetal anoxia in ICP patients and provide a new clinical target for improving adverse fetal outcomes in ICP.

妊娠期肝内胆汁淤积症（ICP）是妊娠特有疾病，可导致难以预测的胎儿宫内缺氧甚至死亡，对胎儿危害极大，其病理机制不清。本课题组前期研究发现ICP胎盘存在缺氧相关的mTOR表达异常。大量研究证实mTORC2-AKT-IP3R信号通路可调控细胞线粒体结合内质网膜（MAMs）中Ca2+转运，实现对内质网和线粒体功能的精确调控，是细胞应对缺氧应激的关键环节。在前期研究结果基础上，本项目拟从胎盘滋养细胞和临床样本两个层面，结合显微观察亚细胞水平Ca2+和核酸蛋白检测的实验方法，阐明胎盘滋养细胞应对缺氧-复氧应激时mTORC2、AKT和IP3R在MAMs的Ca2+转运中的作用和调节机制；揭示ICP胎盘mTORC2-AKT-IP3R的空间动态和表达，及该信号通路介导的MAMs的Ca2+转运状况，为进一步明确ICP胎儿宫内缺氧的病理生理机制提供新的实验依据，为改善ICP胎儿不良结局提供新的靶点和临床切入点。

线粒体结合内质网膜（MAMs）是内质网和线粒体在结构和功能上相连接的重要部分，mTORC2-AKT是调控MAMs功能的核心因子，与IP3R共同作用，精确调控应激状况下细胞MAMs的Ca2+转运，继而决定内质网、线粒体接受应激的强度和细胞命运。本课题组前期研究发现缺氧可抑制胎盘滋养细胞mTOR表达，进一步研究证实，与对照组相比，重度ICP患者胎盘组织p-AKT蛋白水平显著增加；轻、重度ICP患者胎盘组织Rictor蛋白表达水平均显著低于对照组；重度ICP患者胎盘组织IP3R3、p-IP3R3蛋白表达水平均显著低于对照组和轻度ICP组；重度ICP患者胎盘组织IP3R3与p-AKT蛋白共定位显著增加。BeWo细胞中，与常氧组相比，缺氧-复氧组随着复氧时间延长，Rictor、mTOR蛋白水平均呈现先逐渐降低后增加的趋势：缺氧8h组p-AKT蛋白表达水平显著高于常氧组，缺氧-复氧6h组p-AKT蛋白表达较常氧组显著下降，随复氧时间延长，p-AKT蛋白水平逐渐恢复，复氧10h后显著高于常氧组。荧光检测结果显示，与常氧组相比，缺氧-复氧应激下BeWo细胞线粒体与内质网的共定位显著增加、内质网Ca2+降低和线粒体钙Ca2+增加，线粒体ROS增加、膜电位发生去极化，有早期凋亡的趋势，但缺氧-复氧早期细胞仍可代偿恢复这种应激产生的影响。综上，重度ICP胎盘组织中，Rictor低表达和p-AKT高表达将抑制MAMs上IP3R3、p-IP3R3表达，影响经MAMs由内质网向线粒体的Ca2+转运。BeWo细胞应对缺氧-复氧时，mTORC2复合物、p-AKT的表达下调，消减对MAMs上IP3R3的抑制，应激状况下内质网与线粒体耦联区域MAMs的面积增加，内质网释放大量Ca2+至线粒体，内质网储存Ca2+减少，而线粒体Ca2+增加，线粒体接受到应激信号，作出适应性反应。