【作者】 王波；

【导师】 黄荷凤；

【作者基本信息】 浙江大学 ， 妇产科学， 2005， 博士

【摘要】 在生殖、免疫、炎症的一些关键性过程中,分子水平上存在相似的特征。黏附分子L-选择素与其配体的结合介导了免疫系统的淋巴细胞归巢,及炎症过程白细胞与内皮细胞的黏附,并发挥着信号分子作用。近年研究者发现,参与免疫和炎症过程的重要细胞黏附分子L-选择素及其配体也存在于哺乳动物着床期母胎界面上。Genbacev等发现L-选择素与其配体的结合可能构成人类胚胎着床的起始步骤。着床窗口期,胚胎表面的L-选择素和母体子宮内膜L-选择素的寡糖配体似乎都呈现上调趋势。L-选择素配体至少包括四种分子:CD34、粘膜区素细胞粘附分子-1(MAdCAM-1)、依赖糖基化的细胞粘附分子-1(GlyCAM-1)、Sgp200;由于最早发现于小鼠淋巴结高内皮静脉,故将它们统称为周围淋巴结区素(PNAd)。L-选择素识别硫酸化形式的配体,N-乙酰葡萄糖胺-6-转磺酶(GlcNAc6ST)在功能型L-选择素配体生成过程中发挥重要作用。 尽管以往的研究发现了人类母胎界面上存在L-选择素和其配体,但有关早期胚胎发育过程中L-选择素的表达规律、周期性变化的子宮内膜上L-选择素配体表达的时间、空间模式、妊娠建立后它们又将如何变化、它们的表达水平是否与妊娠成功率有关,这一系列问题目前尚未见报道。尽管体外研究表明这对配体一受体确实能够介导细胞之间的黏附,但尚无体内实验证明它们在胚胎着床中的作用。为了深入探讨L-选择素系统在胚胎着床中的功能和影响,我们检测人类母胎界面L-选择素及其配体表达的时间规律、位置特征,分析分泌期子宮内膜L-选择素配体(PNAd)各组成分子以及GlcNAc6ST mRNA水平与体外更多还原

【Abstract】 At the molecular level, there are close links between processes that are key to reproductive, immune, and vascular functions. L-selectin, and its ligands, a pair of important adhesion molecules involved in immune and inflammatory process, have been found on the maternal-fetal interface during mammal implantation. The binding of L-selectin and its ligands, peripheral lymph node addressin (PNAd), mediates lymphocyte homing and adhesion between leukocyte and vascular endothelium. They also function as signal-transducing receptors. Genbacev et al. have found that the binding of L-selectin and its ligands might function as an initiative step in human embryo implantation. During the implantation window, both L-selectin on embryo’s surface and its ligands on endometrium seem to undergo upregulation. Up to date, four glycoprotein ligands have been identified for L-selectin, including CD34, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), and Sgp200 (the latter has not been cloned yet). They were firstly identified on lymph node high endothelial venules of mouse, and collectively termed as peripheral lymph node addressin (PNAd). Sulfatedcarbohydrate structures in ligands are the major domain recognized by L-selectin. N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) functions as a key enzyme in the synthesis of functional ligands for L-selectin.Although L-selectin and its ligands have been found on maternal-fetal interface, there are few reports about their spatial and temporal expression pattern during normal menstrual cycle and early pregnancy. Whether their expression level is associated with successful implantation? Whether they have functional importance in implantation in vivo? These questions are still need to be clarified.In this study, we estimate the expression characteristics of L-selectin on human peri-implantation embryos and its ligands on endometrium throughout the menstrual cycle and during the first trimester of pregnancy. To explore their effect on implantation, we compare the L-selectin ligands (PNAd) and GlcNAc6ST mRNA expression level in secretory phase endometrium between pregnant and nonpregnant patients who underwent in vitro fertilization and embryo transfer (IVF-ET). On the basis of this, we investigate the importance of L-selectin in mouse embryo implantation in vivo.Part One: L-Selectin and Its Ligands (PNAd) Expression on Human Maternal-Fetal InterfaceObjective: To explore the distribution of L-selectin in human peri-implantation embryo and chorion during the first trimester and investigate the spatial and temporal expression of L-selectin ligands (PNAd) in endometrium and deciduas.Methods: (1) L-selectin expression was examined by immunofluorescence in 20 peri-implantation embryos from IVF-ET program (morula, n=8; blastocyst, n=12) and 5 cases of chorion during the first trimester from pregnant women whounderwent induced abortion without medical indications. (2) Immunohistochemistry and Western Blotting were used to examine PNAd expression in endometrium from 76 women (12 from healthy women, in proliferative phase; 64 from sterile women suffering from tubal or male factor, among them, 28 in early-secretory, 36 in mid-secretory phase) and deciduas from 13 women during the first trimester of pregnancy.Results: (1) L-selectin was expressed in membrane and cytoplasma of morula (8/8), blastocyst (12/12, including inner cell mass and trophoblast) and chorion (5/5) in the first trimester, and no significant difference was observed among the above three stages (P>0.05). (2) PNAd localized to the membrane and cytoplasm of luminal and glandular epithelia. Staining was patchy and much less intense during the proliferative phase and deciduas than the secretory phase. In Western Blotting of PNAd, four bands appeared, which were Sgp200, CD34, MAdCAM-1, and GlyCAM-1 respectively, and each was positively correlated with the others significantly (r=0.340~0.718, .P<0.05). The former three molecule levels were significantly higher during secreto更多还原