臺灣博碩士論文加值系統

生殖年齡的女性大約有10-15%會得到子宮內膜異位症，患者通常伴隨慢性腹腔發炎、腹腔疼痛，嚴重者甚至會導致不孕。早在1860年代，即有學者提出子宮內膜異位症，但是到目前為止，詳細的致病機轉仍不清楚。之前的研究指出，前列腺素E2在子宮內膜異位症的生成扮演相當重要的角色。我們實驗室之前證實活化的有絲分裂活化蛋白質激酶會促使環氧化酶二型表現量增加，進而促進前列腺素E2的大量生成。雙特異性去磷酸酶藉由去磷酸化作用抑制有絲分裂活化蛋白質激酶的活性。本篇研究發現，異位的子宮內膜組織的雙特異性去磷酸酶4的表現量較原位的子宮內膜組織來得低。而降低的雙特異性去磷酸酶4則無法抑制ERK的過度活化，並且使得環氧化酶二型大量表現。我們將雙特異性去磷酸酶4的質體轉殖進入異位子宮內膜基質細胞，以探討雙特異性去磷酸酶4對於環氧酵素二型的調控。由實驗結果得知，在異位的基質細胞中，雙特異性去磷酸酶4會降低ERK的活性，並且降低環氧化酶二型的基礎表現量。此外，本實驗也證實將子宮內膜細胞處理缺氧，會抑制雙特異性去磷酸酶4的轉錄，使其不表現。綜合本實驗之結果，缺氧會降低雙特異性去磷酸酶4的表現，使得有絲分裂活化蛋白質激酶不正常的大量活化，導致環氧酵素二型大量表現。這樣的結果暗示缺氧抑制雙特異性去磷酸酶4的表現在子宮內膜異位症的發展中扮演很重要的角色。

Endometriosis affects about 10-15% women of reproductive age, resulting in chronic pelvic inflammation, abdominal pain, and infertility. Endometriosis was first described in 1860. However, the etiology and pathogenesis remain unclear. Prostaglandin E2 (PGE2) is the master regulator that promotes endometriosis formation and causes severe pathological symptoms. We have previously demonstrated that high PGE2 is produced by upregulation of cyclooxygenase-2 (COX-2) in endometriotic stromal cells through activation of mitogen-activated protein kinase (MAPK) pathway. Dual specificity phosphatases (DUSPs) are downstream phosphatases of MAPKs, which act to terminate the activity of MAPKs. Here we showed that expression of DUSP4 in endometriotic tissue was lower than that in normal tissue. Reduced expression of DUSP4 was correlated with aberrant phosphorylation of ERK in endometriotic stromal cells and overexpression of COX-2. To further investigate the effect of DUSP4 on COX-2 expression, we cloned full-length human DUSP4 cDNA and overexpressed it in cells. Results demonstrated that overexpression of DUSP4 reduced the levels of phosphorylated ERK, and ultimately resulted in reduced COX-2 expression. Finally, we showed that reduced expression of DUSP4 in endometriotic cells was mediated by hypoxia-induced transcriptional suppression. Taken together, we provide compelling evidence to demonstrate that hypoxia inhibits DUSP4 expression leading to aberrant activation of ERK, which results in overexpression of COX-2. Our data indicate that hypoxia-inhibited DUSP4 expression is a critical factor for the development of endometriosis.

中文摘要 2
Abstract 3
誌謝 4
緒論 8
材料與方法 15
子宮內膜異位症及正常子宮內膜病人檢體之收集與培養 15
萃取組織核醣核酸 (RNA) 16
質體DNA製備 17
DNA轉殖 ( transfection) 18
缺氧處理 18
萃取細胞核/質蛋白質 19
萃取細胞核醣核酸 19
反轉錄聚合酶連鎖反應 (Reverse transcription polymerase chain reation, RT-PCR)及聚合酶連鎖反應 (polymerase chain reation, PCR) 20
蛋白質濃度分析 ( Lowry assay) 21
西方轉漬法 (Western blot) 21
統計分析 22
結果 23
在患有子宮內膜異位症婦女的異位子宮內膜組織其雙特異性去磷酸酶4 表現量較健康婦女的子宮內膜組織低 23
在患有子宮內膜異位症婦女的異位子宮內膜組織其雙特異性去磷酸酶4 表現量較原位的子宮內膜組織低 23

在患有子宮內膜異位症婦女的異位子宮內膜基質細胞其雙特異性去磷酸酶4 表現量較原位的子宮內膜基質細胞低 24
在293FT細胞株之中，雙特異性去磷酸酶4可抑制ERK和p38之活性 24
在患有子宮內膜異位症婦女的異位子宮內膜基質細胞之中，雙特異性去磷酸酶4可抑制ERK活性 25
缺氧會抑制原位的子宮內膜基質細胞中雙特異性去磷酸酶4的mRNA表現量 25
雙特異性去磷酸酶4調控異位子宮內膜基質細胞之中環氧化酶二型的基礎表現量 26
討論 27
附圖 31
參考文獻 39
附錄 43
細胞培養相關溶液 43
蛋白質相關溶液 44
細胞萃取相關溶液 48
抽取質體 DNA 相關溶液 49
藥品來源 50

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