GnT-Ⅴ调控α5β1整合素的糖基化在子痫前期发病中的作用机制研究

Pre-eclampsia (PE) is a fatal pregnancy complication with multisystem disorders characterized by the onset of hypertension and proteinuria beyond 20 weeks of gestation. Although numerous studies have attempted to elucidate this complex multifactorial disease, its exact etiopathogenesis is still an open question. Now abnormal trophoblastic invasion and spiral arteriolar remodelling is thought to be the primary pathogenesis of PE, in this process glycosyltransferase and protein glycosylation modification play an important role, but its mechanism is still not clear. Our previous studies found that GnT-Ⅴexpressed significant stronger in PE placenta, so to explore its role in PE from the molecular level, we use N-acetylglucosaminetransferase Ⅴ as the breakthrough point, to study its change trend and glycosylation of α5β1 integrin in PE. Through down-regulation of GnT-Ⅴ expression by small interfering RNA or up-regulation of GnT-Ⅴ by plasmid transfection in cell line, using technologies such as qPCR, western blot, L4-PHA blot, immune coprecipitation technology, flow cytometry and transwell chamber etc., we will further test GnT-Ⅴ, α5β1 integrin expression and its glycosylation modification in this two states of cells, and the related cell signal transduction pathway. We will verify these in cell co-culture model and human villous explant culture model. It aimed to illustrate the role of GnT-Ⅴ through controlling the functional protein N-glycosylated in the occurrence and development of PE, and it will provide new ideas for diagnosis and treatment of ischemic placenta diseases, just like PE.

胎盘滋养细胞浅着床和螺旋动脉重铸异常是子痫前期发病的中心环节，糖基转移酶和蛋白质糖基化修饰在该过程中起着重要的作用，但机制尚不清楚。本项目在前期工作的基础上以N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-Ⅴ)为切入点，研究其在子痫前期中的变化趋势和对重要糖蛋白质α5β1整合素的修饰情况；建立GnT-Ⅴ基因沉默和过表达细胞模型，在常氧或缺氧/复氧条件下培养，运用qPCR、WB、L4-PHA blot、IP和Transwell等技术检测细胞中GnT-Ⅴ对α5β1整合素上β1,6-GlcNAc糖支链表达的影响及其调控滋养细胞侵袭和血管内皮细胞损伤的具体机制和相关信号通路，并在细胞共培养和绒毛外植体培养模型上进行验证，从分子水平上阐明GnT-Ⅴ调控α5β1整合素的糖基化参与子痫前期发生发展的机制。该项目的实施将明确子痫前期发病过程中涉及的部分糖生物学机制，为子痫前期等胎盘缺血性疾病的诊治提供新思路。

胎盘滋养细胞浅着床和螺旋动脉重铸异常是子痫前期发病的中心环节，糖基转移酶和蛋白质糖基化修饰在该过程中起着重要的作用，但机制尚不清楚。本项目在前期工作的基础上以N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-Ⅴ)为切入点，我们不仅研究了其子痫前期发病机制也研究了其在调控滋养细胞功能中的作用。我们通过细胞学实验和早孕绒毛外植体等发现（1）GnT-V作为一个负性调节因子在滋养细胞侵袭和早期胎盘发育过程中发挥着重要的作用，这可能是通过直接或间接地调控基质金属蛋白酶MMP2和MMP9的活性来实现的。（2）在绒毛外滋养细胞中存在着这样一条信号通路：氧化应激损伤—GnT-V↑—ERK磷酸化↑—整合素糖基化作用改变-滋养细胞功能损伤，证实了GnT-V通过FAK-ERK信号通路参与子痫前期胎盘形成过程中的滋养细胞侵袭和血管重铸。（3）GnT-V的shRNA和ERK特异性抑制剂PD98059可促进H/R下滋养细胞的迁移、侵袭能力及血管生成能力，从而改善子痫前期胎盘浅着床和螺旋动脉重铸异常情况，为子痫前期的治疗提供了新的思路。