粒線體雌激素受體?瓟梇惜l宮內膜細胞的細胞保護作用

子宮內膜異位症是女性常見的疾病，並已被證實是女性不孕有關。迄今，子宮內膜異位症被認為是一種因為荷爾蒙不平衡所引起的疾病，但其真正的致病機制仍未知。子宮內膜異位生長被證實需要高量的雌激素。雌激素被證實可以刺激粒線體呼吸鏈蛋白質的增加，並且影響粒線體DNA的生合成。我們推測粒線體ERβ可以維護並調節粒線體功能，供應子宮內膜異位生長所需，進而增進細胞的存活。在本研究中，我們發現子宮內膜異位症及子宮腺肌症異位處的內膜細胞中，粒線體ERβ含量比正常的子宮內膜組織多出約2.5倍，同時於病灶組織發現粒線體DNA拷貝數(mitochondrial DNA copy number)增加為對照組的3倍。進而推測粒線體雌激素受體?珗鴾l宮內膜細胞(Hec-1A)有細胞保護的作用。我們以diarylpropionitrile (DPN，一種雌激素ERβ agonist)處理子宮內膜細胞，發現DPN可以保護並減緩細胞受粒線體ATP synthase抑制劑oligomycin或粒線體去偶合劑(uncoupler, carbonyl cyanide m-chorophylhydrazone, CCCP)刺激後所引發的粒線體功能受損及細胞凋亡，都可將細胞存活率恢復至50%。我們發現DPN可以減緩oligomycin 或CCCP所造成的氧化性傷害(oxidative damages)及ATP減量，並維持粒線體膜電位(mitochondrial membrane potential)。我們進一步觀察利用共軛焦螢光顯微鏡觀察到DPN會使ERβ轉位至粒線體並且造成粒線體型態的改變，使細胞呈現大型網狀粒線體分佈，並且可減緩由oligomycin引發的粒線體fragmentation。而DPN的添加處理可增加1.3倍的粒線體DNA拷貝數和mRNA的轉譯效能。此外，我們進一步以粒線體基因免疫沉澱技術(mtDNA immunoprecipitation assay)檢測ER?珙O否與粒線體基因結合，結果顯示ER?珨P粒線體基因D-loop可產生結合，在子宮內膜異位症患者組織中ER?珨P粒線體基因D-loop區域的結合量是對照組的2.6倍；在體外內膜細胞中發現ER??-D-loop的結合量於DPN添加後則增加為對照組的1.3倍。因此我們推測粒線體ERβ可能經由結合到粒線體DNA的D-loop區域進而調控粒線體生合成並提升粒線體功能，進而幫助細胞對抗細胞凋亡。藉由釐清粒線體ERβ在子宮內膜異位症所扮演的角色，希望可作為治療子宮內膜異位症的藥物治療的理論基礎，也可作為未來發展以粒線體ER-β為主要治療標的新子宮內膜異位治療方式之參考。

關鍵字

子宮內膜異位症；雌激素受體；粒線體

並列摘要

Endometriosis is a common disorder presented with infertility, and often affects young women in the childbearing age. However, the precise etiology of endometriosis remains elusive. It is generally considered secondary to the hormonal imbalance. Recently, mitochondrial estrogen receptor (mtERβ) was found in several tissues and the putative function is suggested to enhance respiratory chain function of mitochondria. We hypothesize that mtERβ promotes the endometriotic cell survival by enhancing mitochondrial respiration capacity and changing its susceptibility to apoptosis. In this study, we found that the proportion of ERβ inside mitochondria and the mtDNA copy number were 2.5 times and 3 times increased in the endometriotic tissues. Diarylpropionitrile (DPN, an mtERβ agonist) could attenuate the oligomycin (inhibitor of ATP synthase) or mitochondrial uncoupler (carbonyl cyanide m-chorophylhydrazone, CCCP)-induced mitochondrial dysfunction and cell death in the endometriotic cells. The cell viability was maintained to 50% in the cells co-treated DPN and oligomycin or CCCP. MtERβ was found to regulate mitochondrial morphology (fusion and fission). The predominantly large tubular form was observed in the DPN supplemented cells. It was also demonstrated to attenuate the proportion of fragmented mitochondria followed oligomycin treatment. DPN at 10 nM could reduce the generation of reactive oxygen species (ROS) and ATP depletion in the oligomycin-treated Hec-1A cells, maintain mitochondrial membrane potential, regulate mitochondrial biogenesis, increase 1.3 times mtDNA copy number and mRNA transcripts, and decrease cell apoptosis. MtERβ-mediated maintenance of mitochondrial function was suggested to attenuate cell apoptosis induced by proapoptotic stimuli. Furthermore, the interaction of mtERβ and mtDNA was found to be augmented in the endometriotic tisssue. The mitochondrial DNA immunoprecipitation (mtDNAIP)-PCR method was performed to examine the mtERβ and mtDNA interaction. There were 2.6 times or 1.3 times increase augmented interaction found in the endometriotic tissue from the patieuts with adenomyosis or endometriotic cells with treatment of DPN, respectively. The results will unravel the causal role of mtERβ in the regulation of endometriotic cell growth, which may promote the development of mtERβ directed therapies for patients with endometriosis.

並列關鍵字

endometriosis ； estrogen receptor beta(ER beta) ； mitochondria

參考文獻

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粒線體雌激素受體?瓟梇惜l宮內膜細胞的細胞保護作用

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