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研究生:郭瓊茵
研究生(外文):Chiung-Yin Kuo
論文名稱:過度活化的STAT蛋白在子宮內膜異位症中對前列腺素G/H合成酶-2轉錄調控的探討
論文名稱(外文):Effects of hyperactivated STATs on transcriptional regulation of COX-2 in endometriosis
指導教授:蔡少正
指導教授(外文):Shaw-Jeng Tsai
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:子宮內膜異位症前列腺素G/H 合成酶-2
外文關鍵詞:COX-2endometriosisSTAT
相關次數:
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  • 下載下載:43
  • 收藏至我的研究室書目清單書目收藏:0
當子宮內膜組織在子宮腔以外的位置附著生長即稱為子宮內膜異位症。在生育年齡的婦女中約有10% 罹患子宮內膜異位症,是一種十分常見的婦科疾病。然而到目前為止此疾病的詳細機制尚未被釐清。先前的研究發現前列腺素E2會促進子宮內膜異位基質細胞中雌二醇的產生,而控制前列腺素E2生合成的速率決定步驟是由前列腺素G/H合成酶-2所調控。本實驗室先前的研究已經證實,由於前列腺素G/H合成酶-2驅動子的異常調控使得子宮內膜異位基質細胞中前列腺素G/H合成酶-2表現量會高於正常子宮內膜基質細胞。為了研究在子宮內膜異位基質細胞中前列腺素G/H合成酶-2驅動子的調控機制,我們發現在前列腺素G/H合成酶-2驅動子中有兩個GAS motif,它是一段可讓轉錄活化因子STAT家族蛋白所結合的DNA序列。本論文主要想探討在子宮內膜異位症中STAT蛋白是否會調控前列腺素G/H合成酶-2驅動子。結果顯示STAT1及STAT3在子宮內膜異位組織的磷酸化程度高於正位的子宮內膜組織。為了進一步研究過度活化的STAT蛋白在子宮內膜異位症所扮演的角色為何,使用介白素-1beta刺激同一病人的子宮內膜異位及正位基質細胞後發現,介白素-1beta皆可刺激子宮內膜異位及正位基質細胞中STAT1及STAT3的磷酸化增加,而STAT5的磷酸化則是表現量很低或無法偵測。而STAT3的磷酸化表現在子宮內膜異位基質細胞會比正位基質細胞來的高。另一方面,使用RNA干擾實驗探討STAT1及STAT3蛋白對前列腺素G/H合成酶-2表現的影響發現,無論是單獨抑制STAT1或STAT3,或者同時抑制STAT1及STAT3蛋白質的表現後都無法影響介白素-1beta刺激前列腺素G/H合成酶-2的表現量。相較之下,當使用抑制劑阻礙NF-kB、p38、及ERK訊息傳導路徑的活化時,幾乎可以完全抑制介白素-1beta所刺激的前列腺素G/H合成酶-2表現。從以上結果可知, STAT1及STAT3可能並不參與在介白素-1beta刺激前列腺素G/H合成酶-2表現的訊息傳導路徑中。而子宮內膜異位組織中過度活化的STAT1及STAT3可能是藉由調控其他基因的轉錄活化過程而促進子宮內膜異位症形成。
Endometriosis is defined as the presence of endometrial tissues outside the uterine cavity. It is a common disease that occurs primarily in women of reproductive age and the detailed mechanism is still unclear. Prostaglandin E2 has been shown to stimulate steroidogenesis in ectopic stromal cells and its biosynthesis is controlled by the rate-limiting enzyme termed cyclooxygenase (COX). Previously, we demonstrated that COX-2 expression is greater in ectopic than eutopic stromal cells due to upregulation of COX-2 promoter activity. To delineate the molecular mechanisms responsible for overexpression of COX-2 in ectopic endometriotic cells, we identified two putative gamma-activated sites (GAS) in COX-2 promoter. Signal transducers and activators of transcription (STATs) are transcription factors that bind to GAS motif in the promoter region of target genes. The aim of this project is to investigate the effects of STATs on COX-2 promoter in endometriosis. Therefore, clinical tissues were collected for detecting STATs expression and the data showed that the levels of phospho-STAT1, and phospho-STAT3 are more abundant in ectopic tissue of patients with endometriosis. In order to investigate the role of hyperactivated STATs in endometriosis, paired eutopic and ectopic endometrial stromal cells were treated with IL-1beta and expression of COX-2 was determined. Treatment of endometriotic stromal cells with IL-1beta induced phosphorylation of STAT3 and STAT1 whereas that of STAT5 was undetectable. The levels of phospho-STAT3 were more abundant in ectopic stromal cells than their eutopic counterparts. On the other hand, RNA interference was used to investigate the importance of STAT1 and STAT3 in regulation of COX-2. Ablation of STAT1, STAT3, or both by RNAi did not affect COX-2 expression induced by IL-1beta. In contrast, inhibition of other signaling pathways such as NF-kB, p38, and ERK completely blocked IL-1beta-induced COX-2 expression. These results indicated STAT1 and STAT3 may not be involved in IL-1beta-induced COX-2 expression. We conclude that the hyperactive STAT1 and STAT3 might influence other genes involved in pathogenesis of endometriosis.
目錄...................................................... 1
圖錄...................................................... 4
中文摘要.................................................. 6
Abstract.................................................. 8
緒論...................................................... 10
實驗材料與方法............................................ 23
材料:................................................. 23
子宮內膜異位症病人檢體之收集....................... 23
實驗方法:... ........................................ 23
子宮內膜異位症病人之異位與正位組織保存處理......... 23
子宮內膜異位症病人之異位與正位組織蛋白質抽取....... 23
自正常子宮內膜與子宮內膜異位組織中分離出基質細胞... 24
萃取細胞蛋白質..................................... 25
RNA干擾(RNA interference)........................ 25
蛋白質濃度分析 (Lowry assay)........................ 26
西方轉漬法 (Western blotting)....................... 27
製備小量質體DNA (Minipreparation of plasmid DNA).... 28
製備大量質體DNA (Midipreparation of plasmid DNA).... 29
細胞轉殖 (Transient transfection)................... 30
螢光酵素及Beta-半乳糖苷酶檢測(Luciferase and beta-galactosidase
assay) ................................................32
統計分析 (Statistical analysis)..................... 32
結果 .................................................... 33
STAT1與STAT3在子宮內膜異位症病人的異位組織中活化程度
高於正常子宮內膜組織………………………………………… 33
介白素-1β刺激正常子宮內膜基質細胞STAT1及STAT3磷酸化
的時間點............................................. 34
介白素-1β刺激子宮內膜異位基質細胞後,STAT1及STAT3磷
酸化的時間點......................................... 34
介白素-1β刺激正常子宮內膜基質細胞後,STAT1及STAT3的
磷酸化程度並不會在更短時間點內變化................... 35
介白素-1β刺激子宮內膜異位基質細胞後,STAT1及STAT3的
磷酸化情形同樣不會在更短時間點內有週期性起伏現象..... 36
同一位病人其STAT1及STAT3的磷酸化程度在子宮內膜異位基
質細胞比正常子宮內膜基質細胞高....................... 36
在子宮內膜異位基質細胞中單獨抑制STAT1或STAT3蛋白的表
現不會影響前列腺素G/H合成酶-2的表現................. 37
在子宮內膜異位基質細胞中同時抑制STAT1和STAT3蛋白的表
現不會影響前列腺素G/H合成酶-2的表現................. 37
在正常子宮內膜基質細胞中,介白素-1β刺激後,透過NF-kB、
MAPK及STAT的訊息傳導路徑影響前列腺素G/H合成酶-2的表
現量............................................... 38
在子宮內膜異位基質細胞中,介白素-1β刺激前列腺素G/H合
成酶-2的傳導路徑主要透過NF-kB、MAPK而非STAT蛋白的訊
息傳導路徑........................................... 39
在正常子宮內膜基質細胞中GAS motif對於影響介白素-1β刺
激前列腺素G/H合成酶-2驅動子的活性無統計性差異....... 39
討論 .................................................... 54
參考文獻 ................................................ 61
附錄: ................................................... 66
(一)組織蛋白質來源的子宮內膜異位症病人臨床資料與分期 66
(二)含各種前列腺素G/H合成酶-2驅動子之質體DNA列表….. 67
(三)溶液的配製………………………………………………… 68
細胞培養相關溶液.................................. 68
萃取細胞蛋白質相關溶液............................ 69
蛋白質分析相關溶液................................ 70
質體製備相關溶液.................................. 73
其他.............................................. 74
(四)使用藥品廠牌一覽表 ............................. 75
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