2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645258摘要：胚胎著床於母體的子宮內膜是哺乳類動物傳宗接代中不可或缺的步驟，然而，臨床上體外受精（試管嬰兒）療程卻僅能達到約10-20%的胚胎著床率。依序而言，胚胎著床包括胚胎接近、貼附、及嵌入子宮內膜的過程。在此過程中需要多種細胞素、細胞表面固著分子、及免疫系統共同參與。其中，文獻中曾被報告與胚胎著床相關之細胞表面固著分子包括Integrin α4β1、Integrin αVβ3、MUC-1、E-cadherin、L-selectin 及Trophinin。體外受精週期中，學者們普遍認為高濃度雌激素可能導致子宮內膜接受胚胎著床的機率降低。曾有報告指出可能是高濃度雌激素刺激後，造成子宮內膜組織IL-11 及IL-6 表現量降低、基因表現改變、及DKK1 基因異常等，但詳細機轉目前仍不清楚。本計畫首先將建立體外細胞共同培養模式來模擬胚胎著床。我們把BeWo 細胞株培養成球形之擬似胚胎，將其與不同濃度雌激素刺激後之子宮內膜腺體細胞共同培養，再計算各組之胚胎貼附比率。本研究先以子宮內膜腺體細胞株RL95-2 做實驗，在實驗條件穩定後再以人體細胞接續實驗。之後，我們利用多種實驗方法偵測前述各種細胞表面固著分子在不同濃度雌激素刺激後，於子宮內膜腺體細胞之表現。實驗方法包括：西方墨點法、流式細胞儀、免疫細胞染色、酵素免疫分析、細胞凋亡及週期分析、凋亡體偵測、基質金屬蛋白酵素蛋白質晶片、即時定量反轉錄聚合酶連鎖反應、啟動子冷光偵測、RNA 干擾、以及老鼠體內實驗。藉由一系列實驗之後，希望更加瞭解雌激素作用後子宮內膜腺體細胞中細胞表面固著分子之表現。以此為基礎，我們將逐步探索胚胎著床奧秘，掀開其神秘面紗，期盼未來能夠提高胚胎著床率，造福廣大不孕族群。<br> Abstract: Implantation of the blastocyst into the maternal uterus is a crucial step inmammalian reproduction. In the treatment for infertile couples, however, the lowembryo implantation rate (around 10-20%) is a big obstacle that needs to beovercome. The process of embryo implantation encompasses several distinct stages:apposition, adhesion, penetration, and trophoblast invasion. Multiple systems,including cytokines, adhesion molecules, and immune cells, cooperate closely toestablish a supportive environment for implantation. Several adhesion molecules,including E-cadherin, Integrin α4β1, Integrin αVβ3, L-selectin, trophinin, andMUC-1, have been found to play significant roles during mammalian embryoimplantation.In in vitro fertilization, high estrogen concentration has been found to bedetrimental to reproductive outcome due to its negative effect on the endometrium,although the underlying mechanisms remain unclarified. Reduced expression ofIL-11 and IL-6, altered gene expression profiles, and aberrant DKK1 gene expressionin peri-implantation endometrium might partly account for the impaired embryoattachment for women with extremely high concentration of estradiol around embryoimplantation.In this project, we start with setting up a model that is available for in vitroevaluation of embryo implantation. Pseudo-embryos will be produced from BeWocells, a trophoblast cell line. After that, we will compare the embryo implantationpotentials of trophoblast spheroids on the endometrial glandular cells that have beenpre-treated with different concentrations of estradiol. We then examine the expressionof various adhesion molecules, including E-cadherin, L-selectin, MUC-1, IntegrinαVβ3, Integrin α4β1, and trophinin. The methods for experimental determinationinclude: western blotting, immunocytochemistry, flowcytometry, enzymeimmunoassay, apoptosis and cell cycle analysis, apoptotic body determination, MMPprotein array, real-time quantitative RT-PCR, promoter reporter assay, RNAinterference, and knockout mouse model.The aim of this project is to investigate the effect of different concentrations ofestrogen on the embryo implantation potential. We also try to clarify the molecularmechanism of estrogen-induced alteration on endometrial glandular cells. Resultsobtained from this project may provide important information for the clinicallyrelevant role in enhancing embryo implantation.胚胎著床高濃度雌激素細胞凋亡embryo implantationhigh estrogen concentrationapoptosis