粒線體p53及雌激素受體在調控光照傷害之細胞保護功能

Mitochondria p53 and ER mediated cytoprotection in rhodamine123 mediated photodamage

翁顤珊(Yao-Shan Weng)

指導教授 ： 高淑慧

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00018

摘要

雌激素調節子宮內膜生長，分化以及修復。其受體又分α、β兩種型態。在子宮內膜異位細胞中，有研究指出其雌激素受體(estrogen receptor, ER)與p53會有增加的趨勢。已知p53在細胞遭受壓力時會移動到粒線體中參與細胞凋亡或修復作用，然而，這兩者之間是否有關連性，尚未完全釐清 。 所以本論文擬探討子宮內膜細胞中ER與p53的相關性。本研究利用Rhodamine 123專一標定粒線體，利用光照傷害(photodamage) 粒線體來探討p53與ER的關連性。研究發現，將Hec-1A細胞株在光照傷害後給予 diarylpropionitrile (DPN，雌激素ERβ agonist)以及 propyl pyrazole triol (PPT，雌激素ERα agonist) ，會降低光照傷害造成粒線體受損導致的細胞死亡。並且發現加入DPN後，細胞的生長速率有顯著上升的情形。此外，在光照傷害及加入DPN或PPT作用，皆不會影響p53總蛋白的表現量，表示光照傷害為細胞可修復性，因此p53並不會大量表現使細胞進行凋亡作用。接著我們使用粒線體分離技術來觀察p53在粒線體中表現的情形，結果發現單純光照傷害不會增加p53進入粒線體中的趨勢，然而加入E2後其粒線體中p53的表現顯著增加。我們又進一步利用粒線體基因免疫沉澱技術(mtDNA immunoprecipitation assay)檢測在光照傷害下p53是否與粒線體基因結合，結果顯示p53可與粒線體基因產生結合，並且發現在添加E2、DPN及PPT後，p53與粒線體DNA結合的表現量分別增加了2.3倍、1.54倍以及4.15倍。此外我們也分析在光照傷害後加入DPN以及PPT的組別中，其Reactive oxygen species(ROS)及 8-hydroxy-2’-deoxyguanosine (8-OHdG)表現量有顯著下降的趨勢。接著我們蒐集子宮內膜異位症腹膜液中的異位內膜細胞，並且比較嚴重程度及是否使用Gonadotropin releasing hormone analogue (GnRHa)治療；結果顯示p53表現量具有差異性。發現徵狀越輕微的病人細胞，表現較多正常片段的p53 mRNA，在蛋白質方面則以p53 tetramer的表現量較為顯著。因此我們推測，p53 可能經由Estrogen刺激後轉位至粒線體中進而與粒線體DNA結合進行修復或保護粒線體DNA並增加抗氧化酵素的產生，幫助細胞對抗ROS的傷害。藉由釐清p53與ER的關連性以及在子宮內膜異位症患者中引發的機轉，希望可以作為未來新的方向思考治療子宮內膜異位症以及癌症的生成。

關鍵字

p53 ； 雌激素 ； 雌激素受體 ； 子宮內膜異位症

並列摘要

Estrogen is required to induce a full uterine proliferation, differentiation, and repair of endometrial surface. Estrogen has cell-specific effects on a variety of physiological condition including of mitochondrial biogenesis and activity mediated by estrogen receptor alpha and beta (ER-α and ER-β). Recent studies indicate that ER and p53 increased in endometriotic tissues. p53 is demonstrated to move into mitochondria and involve in regulating cell apoptosis or DNA damage repair when cell suffer from stress. However, the relationship between the ER and p53, are not fully clarified. We hypothesized that estrogen promotes the endometrial cell survival by reducing reactive oxygen species, enhancing p53 checking efficiency, and changing mitochondrial susceptibility to apoptosis. To test this hypothesis, we used the mitochondria-specific-targeting Rhodamine 123 mediated mitochondrial photodamage treatment, and ERα-specific agonist propyl pyrazole triol (PPT) and ERβ-specific agonist diarylpropionitrile (DPN) to address the relationship between the ER and p53. We found that when the treatment with estradiol, or DPN, or PPT, would rescue the cells from photodamage-induced mitochondrial damage and cell death. The growth curve was significantly increased in the DPN-treated cells. No significant increase of p53 in the photodamaged cells. However, the p53 in mitochondria was increased after treated with E2. We used mitochondrial DNA immunoprecipitation assay to identify the interaction of p53 and mitochondrial DNA (mtDNA) when cells suffer from photodamage. There were 2.3-, 1.54-, and 4.15-folded increase in p53- mtDNA interaction in the treatment of E2,DPN and PPT, respectively. We further used confocal fluorescence microscopy and flow cytometry to analyze the damages in photodamaged cells. We found the use of DPN or PPT can significantly reduce the generation of reactive oxygen species (ROS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) after photodamage treatment. Additionally, the primary endometriotic cells were obtained from ectopic endometrial peritoneal fluids to address the alteration of p53 expression profile. We found the cells harbored more normal p53 mRNA segments and the dimmer type of p53 after GnRH agonist treatment. Therefore, we speculate, p53 may be stimulated by estrogen and translocate into mitochondria. P53 will interact with mtDNA to maintain mitochondrial DNA integrity and promote the production of antioxidant enzymes to help the cells against Rhodamine 123 mediated mitochondrial photodamage. The results will unravel the relationship between the ER and p53 in mitochondria, which may promote the new directed therapies of endometriosis and tumor formation.

並列關鍵字

estrogen ； estrogen receptor (ER) ； endometriosis ； p53

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