2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660221摘要：胚胎著床過程涵蓋了一連串複雜的分子及細胞互動，而且特定時間需要特定分子參與。伴隨著胚胎發育，子宮內膜也同步進行細胞增殖與轉化。此過程乃藉由雌激素與黃體素作用，讓子宮內膜轉化為可以接受胚胎著床的狀態。體外受精週期中，普遍使用大量排卵針劑刺激產生更多卵子，但同時也在體內製造高濃度雌激素。許多研究均認為高濃度雌激素可能導致子宮內膜接受胚胎著床機率降低，但詳細機轉目前仍不清楚。 我們已在小鼠動物實驗中發現卵巢過度刺激誘發大量排卵時，胚胎活產率顯著降低。本計畫中，我們將運用我們已經建立的動物模式，配合蛋白質體學分析、生物資訊學、及細胞生物學研究方式，系統性探討高濃度雌激素對子宮內膜組織的效應，來研究高濃度雌激素導致子宮內膜接受胚胎著床機率降低的分子機制。 三年期計畫中，我們的分年計劃目標之研究策略與方法為： 第一年：主要目標是利用蛋白質體學篩選出高濃度雌激素作用於子宮內膜組織的標的分子。研究策略與方法為: 1. 以 PMSG 刺激小鼠大量排卵，確認血中雌激素濃度升高後，在 implantation window (3.5d)，分離子宮內膜組織之上皮與間質細胞。 2. 運用蛋白質體學分離不同雌激素濃度作用後，子宮內膜上皮與間質細胞蛋白質。 3. 運用氣相層析質譜儀(GC-MS)分析實驗組與控制組中具差異性的蛋白質身份，進一步進行蛋白質身份鑑定。 4. 以免疫組織染色，確認由蛋白質體學分析所得之差異性的蛋白質，在子宮內膜組織中的表現。 第二年: 主要目標是以細胞分子生物學進一步驗證，前述由蛋白質體學分析所得之特定蛋白質分子族群，接受高濃度雌激素作用的調控機制。 1. 以小鼠子宮內膜上皮細胞為模型，利用 western blot 驗證特定蛋白質分子族群接受高濃度雌激素刺激後的表現。 2. 以 RTK array 驗證探討高濃度雌激素對小鼠子宮內膜上皮細胞之訊息傳遞影響。 3. 以 decoy ODN 探討高雌激素對小鼠子宮內膜上皮細胞之特定蛋白質分子的轉錄調控。 4. 以訊息傳遞化學抑制劑及基因干擾技術釐清高雌激素影響之 RTK, specific protein, transcriptional factor 之間的關係。 第三年: 主要目標是以基因干擾技術與細胞生物學，探討特定高濃度雌激素作用之基因蛋白質在胚胎著床過程中扮演之角色。 1. 以基因干擾技術抑制小鼠子宮內膜上皮細胞基因蛋白質表現。 2. 利用我們已建立之體外胚胎著床模式，確認特定高濃度雌激素作用之基因蛋白質在小鼠子宮內膜上皮細胞之胚胎著床功能。 我們預期本研究結果將能提供高濃度雌激素干擾子宮內膜上皮細胞，而影響著床之機制。希望充分了解這些機轉後，有助於提高未來人工生殖科技的胚胎著床率，造福廣大不孕族群。 <br> Abstract: Embryo implantation involves a complex succession of genetic and cellular interactions, all of which must be completed within an optimal time frame. Embryo developments are synchronized with endometrial proliferation and differentiation, which is under the effects of estrogen and progesterone that result in a favorable uterine environment for embryo implantation. In in vitro fertilization, multifollicular development is achieved by gonadotropins to increase the number of embryos available for transfer, and ovarian stimulation inevitably causes a high serum estrogen concentration. Reports have revealed that high serum estrogen levels result in lower pregnancy and embryo implantation rates. However, the underlying mechanism is not well known yet. Using a mouse model, we found that live birth rates were apparently decreased after ovarian hyperstimulation. In this project, we plan to investigate the effects of high estrogen concentrations on endometrium. Proteomics, bioinformatics, and cellular biological experiments will be done to systemically explore its underlying mechanism. In the 1st year, we will use proteomics to screen the molecules in endometrium that are differently expressed after the treatment with high estrogen concentrations. Endometrial glandular cells (EGCs) and stromal cells (ESCs) are separated from mouse endometrium, and gas chromatography–mass spectrometry (GC-MS) is employed to identify the differently expressed proteins. Immunohistochemistry is also done to confirm the in vivo expression of these proteins in mouse endometrium. In the 2nd year, western blot is done to verify protein expression of EGCs after high estrogen concentration stimulation. Receptor Tyrosine Kinase (RTK) array is performed to examine the high estrogen-dependent signaling pathway. In addition, decoy oligodeoxynucleotide (ODN) is undertaken to investigate the transcriptional regulation. Signal transduction inhibitors, as well as RNA interference, are used to clarify the relationship among RTK, specific proteins, and transcriptional factors. In the 3rd year, RNA interference will be done to suppress the expression of before-mentioned genes on EGCs. After that, in vitro embryo implantation assay is performed to test the embryo implantation potentials on these gene silenced EGCs under high estrogen stimulation. We expect there would be fruitful results after completion of these experiments. These results might provide important information for the clinically relevant role in enhancing embryo implantation rates for infertile women in the future.胚胎著床高濃度雌激素子宮內膜上皮/間質細胞蛋白質體學embryo implantationhigh estrogen concentrationendometrial glandular/stromal cellproteomics