糖基化依赖性uPA/uPAR分子结合及功能在胚胎植入和相关妊娠疾病中的作用机制研究

Embryo proliferation, implantation and invasion, as well as angiogenesis are the key events to the success of pregnancy. Our research team has been studying reproductive biology on glycobiology for many years. Recently, we got important findings that the expression of urokinase-type plasminogen activator and its receptor (uPA/uPAR) was decreased in abnormal pregnancy patients, and the expression of key enzyme for O-fucosylation on uPA was also downregulated. The invasion and migration abilities of cells were weakened after N-glycosylation sites on uPAR were mutated. Therefore, we propose the novel hypothesis that the O-fucosylation on uPA and N-glycosylation on uPAR are sugar dependent for its combination, activation and functions. Based on the bioinformatics prediction, the study will elucidate the molecular mechanism of uPA/uPAR glycosylation in ligand/receptor binding and activation by expression regulation of enzymes, active sites mutation and glycosylation sites mutation, as well as kinds of protein interaction techniques. The setup of glycoprotein interaction analysis platform will supply the basis for profoundly explore the mechanism and patterns of glycosylation. To observe the roles of glycosylation in embryo implantation, placenta functions and related pregnancy diseases. Meantime, to establish lectin ELISA to detect uPA/uPAR glycosylation in IVF-ET embryo secretion and serum to supply the diagnosis and treatment foundation for clinical practice of reproduction.

胚胎的增殖、植入侵袭和血管重塑能力是决定妊娠成功的关键。课题组多年开展糖生殖生物学研究，近期有重要发现：尿激酶型纤溶酶原激活物及受体（uPA/uPAR）在异常妊娠患者减少，且胚胎细胞中调控uPA的O-岩藻糖基化关键酶表达降低；而uPAR的N-糖基化位点突变使细胞植入能力减弱。因此，课题拟提出并验证uPA的O-岩藻糖基化和uPAR的N-糖基化为二者结合、激活和功能发挥的糖基化依赖性新学说。在生物信息学预测基础上，通过多种酶表达调控、活性位点和糖基化位点突变及蛋白质相互作用技术，解析uPA/uPAR糖基化在配体受体结合和激活中的作用机制；糖蛋白相互作用分析平台，为深入探讨糖基化机制及规律提供条件；观察uPA/uPAR糖基化在胚胎着床、胎盘功能和相关妊娠疾病中的作用，并建立凝集素ELISA检测IVF-ET分泌液及血清uPA/uPAR糖基化的新方法，为临床生殖医学实践提供诊断和治疗依据。

胚胎的增殖、植入侵袭和血管重塑能力是决定妊娠成功的关键。项目得到如下结果：（1） 通过血清学、组织学等临床样品分析，发现uPA，poFUT1在不孕症患者血清中、以及流程患者绒毛中的含量显著降低；（2）通过细胞学实验，阐明uPA的O岩藻糖基化是受体/配体（uPA/uPAR）相互作用的关键；（3） 通过激素或细胞因子/糖合成关键酶/受体配体糖基化网络研究，揭示EREG通过上调uPA上O-岩藻糖基化，促进uPA/uPAR相互作用，介导滋养层细胞植入过程；（4）uPA的O-岩藻糖基化参与子宫蜕膜血管生成过程。课题提出并验证uPA的O-岩藻糖基化为uPA-uPAR二者结合、激活和功能发挥的糖基化依赖性新学说，深入探讨糖基化在胚胎着床中的作用，为临床生殖医学实践提供诊断和治疗依据。