水通道蛋白1介导巨噬细胞极化调控滋养细胞侵袭的机制研究

Normal trophoblast invasion plays a key role in embryo implantation and placenta information. Abnormal trophoblast invasion can result in abortion, pre-eclampsia, fetal growth restriction and placenta implantation. Macrophages at the maternal-fetal interface affect trophoblast invasion and the mechanism is not clear. Our previous study shows aquaporin 1(AQP1) is expressed in trophoblast of placenta, the phenotypes of AQP1-knockout pregnant mice are changed as follows, the conception rate is decreased, the fetal mice growth is restricted, the placental construction is disordered and the osmotic water permeability was reduced. The results suggests AQP1 is involved in trophoblast invasion. Recent study reveals AQP1 affects macrophage polarization and regulates macrophage migration. Therefore, we hypothesize that AQP1 can directly influence trophoblast invasion, and further regulate trophoblast invasion by AQP1-mediated decidual macrophage polarization. We aim to establish the primary AQP1 wild type and knockout mice trophoblast and macrophage cell culture system in vitro, to analyze the macrophage polarization (M1 and M2), to compare the trophoblast invasion in different cells co-culture condition by RTCA. We explore the mechanism of trophoblast invasion by AQP1-mediated macrophage polarization, which will provide a treatment target and theoretical basis for trophoblastic invasion disorder related disease.

正常的滋养细胞侵袭力是胚胎着床、胎盘形成的关键。滋养细胞侵袭力异常可导致流产、子痫前期、胎儿生长受限及胎盘植入等疾病。母胎界面巨噬细胞等免疫细胞影响滋养细胞的侵袭，具体机制不清。课题前期研究显示水通道蛋白1（AQP1）表达于胎盘滋养细胞，AQP1敲除鼠受孕率下降、胎鼠生长受限、滋养细胞排列紊乱且水通透性下降，提示AQP1可能参与了滋养细胞的侵袭。近期发现AQP1影响腹膜巨噬细胞的极化类型，并调节其迁移。因此我们假设：AQP1不仅可直接影响滋养细胞侵袭，而且通过介导蜕膜巨噬细胞极化调控滋养细胞的侵袭。课题将应用AQP1基因敲除鼠，建立原代滋养细胞、巨噬细胞培养体系，分析巨噬细胞M1、M2极化状态，应用实时无标记细胞分析技术（RTCA）研究不同细胞共培养条件下滋养细胞侵袭力的变化，探讨AQP1介导巨噬细胞极化调控滋养细胞侵袭的机制，从而为滋养细胞侵袭力异常相关疾病提供新的治疗靶点和理论依据。

胎盘滋养细胞侵袭力异常可导致流产、子痫前期、胎儿生长受限及胎盘植入等疾病。蜕膜免疫细胞在母胎界面免疫状态的形成和维持中发挥了重要的调控作用，母胎界面免疫细胞如果发生分化异常、亚群平衡失调或功能异常时，会导致滋养细胞侵袭异常，从而出现流产、子痫前期、胎儿生长受限等病理妊娠及不良妊娠结局。大量研究显示水通道蛋白1（Aquaporin 1，AQP1）不仅调控多种细胞的水通透性，而且介导病理生理变化过程中的细胞迁移及侵袭。已有文献报道缺氧可以诱导AQP1 mRNA的表达，组织缺氧的病理状态刺激了AQP1的转录表达，提出缺氧诱导因子1α（hypoxia-inducing factor 1α，HIF-1α）参与AQP1的转录调控。.课题组前期研究发现AQP1敲除鼠的受孕率降低、胎鼠体重降低，胎盘病理提示：滋养细胞排列紊乱、血管生成异常，滋养细胞水通透性下降，提示AQP1可能参与了滋养细胞的侵袭。课题应用siRNA转染技术，通过实时无标记细胞分析技术（RTCA）检测滋养细胞转染AQP1 siRNA后侵袭力、增殖情况明显下降。滋养细胞转染AQP1-siRNA后细胞活性降低。滋养细胞置于低氧环境（1%O2、94%N2、5%CO2）中培养24小时，滋养细胞HIF-1α表达升高，AQP1 表达升高，滋养细胞侵袭力增加。人早孕蜕膜组织原代培养巨噬细胞，蜕膜巨噬细胞上清与滋养细胞共培养24h后，RTCA结果显示蜕膜巨噬细胞分泌的细胞因子促进滋养细胞的侵袭, PCR结果显示滋养细胞MMP9, AQP1 及HIF-1α表达升高。以上研究提示AQP1促进人滋养层细胞系HTR8/Svneo的侵袭、增殖以及细胞活性。低氧情况下，HIF-1α介导AQP1表达升高，从而促进人滋养层细胞系HTR8/SVneo的侵袭力。人早孕蜕膜巨噬细胞在体外培养条件下通过HIF-1α相关通路调控滋养细胞侵袭。