WFS1致妊娠肝内胆汁淤积模型胎鼠缺氧的机制研究

The preterm acute fetal anoxia in intrahepatic cholestasis of pregnancy (ICP) is an important reason of perinatal mortality, which also results in high rates of iatrogenic preterm birth and cesarean delivery. The mechanism of acute fetal anoxia in ICP is unclear. Blood flow regulation of utero-placental-fetal unit in hypoxia stress is the most important mechanism in coping with fetal anoxia in late pregnancy. Previous studies indicated that the corticotropin-releasing hormone (CRH) and urocortin (UCN), the most potent concentration-dependent vasorelaxation factors, were down-regulated in placental tissues of ICP, indicated that the dysregulation of placental stress hormone is related to the dysfunction of endoplasmic reticulum stress in placental tissues and cells. WFS1 is a key gene in regulating the unfold protein response (UPR) and endoplasmic reticulum stress, which may also play a role in regulating the expression of placental stress hormone in ICP. This study use the intrahepatic cholestasis pregnant rat model and technical of gene over-expression/silence to observe the expression and consistency of placental WFS1, UPR-related signaling pathway, stress hormone (CRH/UCN) and fetal hypoxanthine (a factor of acute anoxia). Moreover, it is important to explore the underline mechanism of fetal anoxia, that is WFS1 interfere the synthesis and secretion of placental stress hormone via UPR signaling pathway, thus impair the blood flow stress regulation of utero-placental-fetal unit and lead to acute fetal anoxia. There is no literature about the role of WFS1 and UPR signaling pathway in utero-placental-fetal unit hypoxia stress compensatory and acute fetal anoxia mechanism of ICP. This study will provide new clinical method in preventing and treating acute fetal anoxia and even fetal death in ICP.

妊娠肝内胆汁淤积症(ICP)足月前急性胎儿缺氧是围生儿死亡的重要原因，其机制不清。子宫胎盘胎儿单位缺氧应激血流调节是对抗孕晚期胎儿缺氧的重要机制。前期研究发现ICP胎盘应激激素促肾上腺皮质激素释放激素(CRH)和尿皮素(UCN) 呈低表达，提示可能与其组织细胞内质网应激异常有关。WFS1是调控未折叠蛋白反应(UPR)和内质网应激的重要基因，可能参与ICP胎盘应激激素表达调控。研究拟采用ICP孕鼠及其缺氧模型，通过过表达或沉默WFS1，观察胎盘WFS1、UPR信号通路、应激激素CRH/UCN和胎鼠急性缺氧指标的表达情况和一致性，了解WFS1通过调控UPR信号通路影响胎盘应激激素合成分泌、损害子宫胎盘胎儿单位血流应激调节、导致ICP胎鼠缺氧的可能机制。WFS1和UPR信号通路在子宫胎盘胎儿单位低氧缺氧应激血流调节中的作用尚未见文献报道，本研究可能为防治ICP急性胎儿缺氧提供新的临床方法。

前期研究发现妊娠期肝内胆汁淤积症（ICP）胎盘应激激素促肾上腺皮质激素释放激素(CRH)和尿皮素(UCN) 表达异常，这一现象与内质网应激及其相互关系尚不清楚。.根据项目主要研究内容，采用临床标本、组织病理及动物模型进行研究并取得相应的数据和结果。.一、采用ELISA，qPCR和Western blot观察血浆和胎盘组织WFS1与CRH、UCN表达，发现ICP胎盘组织中WFS1高表达，与CRH、UCN表达呈正相关。.二、采用qPCR和Western blot观察胎盘组织UPR三条通路关键因子（PERK、ATF6、IRE1）表达，发现ICP胎盘组织中PERK高表达，提示PERK通路可能是ICP内质网应激调节关键通路。.三、通过雌激素诱导建立肝内胆汁淤积孕鼠模型，动态观察缺血缺氧再灌注对胎盘组织UPR通路、WFS1、CRH、UCN表达的影响，发现WFS1与内质网应激存在相关，WFS1参与调控胎盘组织CRH、UCN的合成和分泌。UPR通路关键因子表达数据正在分析中。.四、WFS1通过UPR信号通路调节应激激素的表达.本项目原设计采用肝内胆汁淤积孕鼠模型，对WFS1基因进行过表达或沉默，考虑到在动物实验中干扰因素太多，难以排除混杂因素。经课题组成员讨论，决定选用HTR8/SVneo细胞系，采用胆汁酸诱导建立胆汁淤积及缺氧细胞模型，慢病毒沉默WFS1基因，观察WFS1对UPR通路的影响。WFS1敲减后PERK通路关键因子（PERK、ATF4、GRP78）表达升高，表明WFS1可能通过负向反馈调节内质网应激。胆汁淤积及缺氧细胞模型WFS1及PERK通路关键因子表达存在差异，表达规律正验证分析中。模型细胞CRH、UCN表达情况正在实验。.综上：WFS1可能通过负向反馈调控PERK通路，影响ICP胎盘细胞内质网应激，参与调控应激激素CRH、UCN表达，影响ICP胎盘单位应激血流调节。