2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704753摘要：不孕症治療過程導致的血清中高濃度雌激素，臨床上研究顯示可能導致胚胎著床失敗。我們先前的研究顯示高濃度雌激素會藉由增加子宮內膜上皮細胞之IκB-α 表現而影響NF-κB活化，進而抑制抗凋亡蛋白質Bcl-2 的表現而造成細胞凋亡 (發表於J Clin Endocrinol Metab2014)。我們最近的研究成果證明了高濃度雌激素會調控Heat shock factor 1 而增加Hsp70 表現，而Hsp70 會藉由與IKK-g形成複合體而影響NF-κB 活化 (發表於Biol Reprod 2016)。我們認為高濃度雌激素對子宮內膜細胞可能是一種壓力，因此我們使用細胞壓力蛋白質晶片檢測高濃度雌激素作用下，子宮內膜細胞之細胞壓力蛋白表現模式。初步研究結果顯示heat shock proteins、cytochrome c、SOD2 / MnSOD 等數種細胞壓力蛋白會被高濃度雌激素誘發其表現。由於細胞壓力蛋白的調控與粒線體損傷有關，因此在本三年期計畫中我們將利用細胞與活體實驗，探討高濃度雌激素是否引發子宮內膜細胞之粒線體損傷，釐清引發粒線體損傷的分子機制，並進一步探討粒線體相關治療在改善子宮內膜細胞的效應。本計畫的分年目標與執行策略、方法分別為：第一年研究目標是探討高濃度雌激素對粒線體數量與功能變化之效應，執行策略包括內膜細胞(in vitro)實驗與活體動物(in vivo)實驗。體外細胞實驗部分，首先分離出小鼠子宮內膜細胞，初代培養後進行不同濃度之雌激素作用，分析可反映粒線體功能之ATP 製造；利用粒線體特殊染劑JC-1 偵測內膜細胞中單位細胞之粒線體數目，並以粒線體DNA qPCR 方式量化。動物實驗部分，以我們先前建立的高濃度雌激素動物模式，取得內膜檢體。利用粒線體特殊染劑JC-1 偵測內膜細胞中粒線體數目，並以電子顯微觀察粒線體形態變化。第二年研究目標是釐清高濃度雌激素影響粒線體的分子效應。執行策略是以內膜細胞模式，運用訊息傳遞化學抑制劑與抗氧化劑，探討高濃度雌激素調控的訊息傳遞路徑以及氧自由基對於粒線體之調控，並利用qPCR 與EIA 偵測內膜細胞之粒線體數目與活性變化；利用western blot 定量分析粒線體蛋白質變化；並以Seahorse Technology 分析比較高濃度雌激素對內膜細胞粒線體代謝之效應。第三年研究目標是針對受高濃度雌激素干擾粒線體數目與活性的子宮內膜細胞，給予anti-oxidant、ATP、及促進mitochondria synthesis 等粒線體治療方式。執行策略是以體外細胞(in vitro)實驗，測量評估其粒線體數目與活性之回復情形；並以活體動物(in vivo)實驗評估胚胎著床機率之變化。本研究成果將提供粒線體在高濃度雌激素干擾內膜細胞中致病的角色，也能提供以粒線體治療為分子標靶在此類病症的治療潛力。<br> Abstract: High serum estrogen concentrations following ovarian hyperstimulation have been generally foundto lead to reduced embryo implantation potentials in infertile women. Our previous study revealed thathigh estradiol (E2) concentrations result in apoptosis of endometrial epithelial cells (EECs) throughinhibiting Bcl-2 expression by inducing IκB-α expression and inhibiting NF-κB activation (J ClinEndocrinol Metab 2014). Our ensuing study demonstrates that high E2 concentrations enhance HSF-1and accordingly increase Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-gand inhibits pIκB-a, which consequently suppresses NF-κB activation (Biol Reprod 2016).Mitochondria are the energy source of cells, and their healthy status in endometrial cells should behighly correlated with the embryo implantation capability. Reports showed that during cell apoptosis,mitochondria experience a progressive dysfunction. Our preliminary data revealed that high E2concentrations induce cell stress related proteins, such as heat shock proteins, cytochrome c andsuperoxide dismutase 2 (SOD2). They respond by structural adjustment of mitochondria, cell apoptosis,and the initiation of antioxidant functions. These results imply that the high E2-induced endometrial cellapoptosis maybe related to mitochondrial damage. However, this effect has never been investigated.In the 1st year, we use a mouse model to investigate the in vitro and in vivo effects of high E2concentrations on mouse endometrial cells. In in vitro experiments, mouse endometrial cells arepurified, followed by stimulation with different E2 concentrations. ATP production, which stands formitrochondrial function, is measured. The number of mitochondria is determined by mitochondriaspecific florescence dye JC-1, and is quantified by mitochondrial DNA qPCR. In vivo mouseexperiments are also done with female mice injected with different doses of equine chorionicgonadotropin (eCG). The number of mitochondria is determined by JC-1, and the structure ofmitochondria is examined by electron microscope.In the 2nd year, we aim to study the underlying mechanism of high E2-induced mitochondrialdamage in endometrial cells. Specific signaling inhibitors and antioxidant agents are used to investigatethe high E2-induced signaling pathways. Regulation of mitochondria by oxidative stress is also studied.Quantitative PCR and EIA will be done to measure the number and activity of mitochondria inendometrial cells. Western blot is done to determine mitochondrial proteins, and Seahorse Technologyis performed to study the mitochondrial metabolism.In the 3rd year, our goal is to investigate the effect of mitochondrial therapy. Antioxidant agents,ATP, and mitochondria synthesis enhancing agents are added in vitro to mouse endometrial cells thatare stimulated with high E2 concentrations. ATP production, number of mitochondria determined byJC-1 staining, and mitochondrial DNA qPCR will be done to evaluate the number and activity ofmitochondria in endometrial cells after mitochondrial therapy. In vivo mouse embryo implantation rateis also studied with donated embryos transferred into recipient female mice with high serum E2concentrations.We expect there will be fruitful results after completion of these experiments. Results obtainedfrom this project might provide important information with regard to the effect and molecularmechanism of high E2-induced mitochondrial damage in endometrial cells.