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系統識別號 U0026-0808201415405300
論文名稱(中文) 雙特異性去磷酸酶-2參與子宮內膜異位症調控血管新生之角色
論文名稱(英文) Functional Role of Dual Specificity Phosphatase-2 in Angiogenic Process of Endometriosis
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 102
學期 2
出版年 103
研究生(中文) 張寧
研究生(英文) Ning Chang
學號 S36014017
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-蔡少正
共同指導教授-吳孟興
口試委員-蔡英美
口試委員-楊尚訓
中文關鍵字 缺氧  雙特異性磷酸酶-2  介白素-8  細胞外訊息調節激酶  CCAAT/增強子結合蛋白  血管新生  子宮內膜異位症 
英文關鍵字 hypoxia  dual specificity phosphatase-2 (DUSP2)  interleukin-8 (IL-8)  extracellular-signal regulated protein kinase (ERK)  CCAAT/enhancer-binding protein (C/EBP)  angiogenesis  endometriosis 
學科別分類
中文摘要 子宮內膜異位症是最常見的婦科疾病之一,其定義為子宮內膜腺體以及基質組織生長於子宮腔外,其複雜的致病機轉目前仍未能被清楚解釋。目前最被接受的理論之一為婦女月經時剝落的子宮內膜組織碎片隨著經血逆流至腹腔,並於腹腔內生長。當組織進入到腹腔環境時,細胞存活下來最大的挑戰之一是這些異位組織必須克服缺氧的環境壓力。先前的研究結果指出,一種對於細胞外訊息調節激酶具專一性的去磷酸酶─雙特異性去磷酸酶-2─在異位子宮內膜組織中的表現量會被缺氧誘導因子所抑制並促使子宮內膜異位症之進程。過去許多研究已知,缺氧能促發血管新生幫助細胞存活下來,然而雙特異性去磷酸酶-2表現量下降對於血管新生的機制目前尚不清楚。本論文主要探討子宮內膜異位症的發展過程中,透過缺氧所造成的雙特異性去磷酸酶-2表現量下降如何調節血管新生的機制。由全基因體的基因篩選結果顯示,雙特異性去磷酸酶-2調控一群與血管新生有關的基因。即時定量聚合酶鏈鎖反應結果顯示,介白素-8在異位子宮內膜基質細胞的表現量顯著高於原位,此與雙特異性去磷酸酶-2的表現模式呈現負相關。利用原位子宮內膜基質細胞以缺氧處理或以微小干擾核醣核酸減弱雙特異性去磷酸酶-2的表現,都能誘導介白素-8的傳訊者核醣核酸表現顯著增加。透過報導基因測定啟動子活性結果發現,缺氧抑制雙特異性去磷酸酶-2的表現,其下游透過轉錄調控因子CCAAT/增強子結合蛋白α增加介白素-8的表現。進一步利用染色質免疫沉澱定量分析證明,缺氧顯著增加CCAAT/增強子結合蛋白α與介白素-8啟動子的結合。原位子宮內膜基質細胞經過缺氧處理或以微小干擾核醣核酸降低雙特異性去磷酸酶-2表現,收集其培養液處理人類臍靜脈內皮細胞,能顯著促進內皮細胞形成網絡之能力,而利用抑制劑阻斷人類臍靜脈內皮細胞之介白素-8受體,則能顯著抑制其形成網絡之現象。最後,我們以小鼠為實驗模式,利用自體移植手術誘導異位子宮內膜組織病灶形成,證實了給予介白素-8接受器抑制劑處理,除了可以有效減少類子宮內膜異位組織的數目與大小,同時還可以減少組織內微血管的數目。綜合言之,我們發現子宮內膜異位症基質細胞失去雙特異性去磷酸酶-2可以引發介白素-8的表現,並導致異位組織可以促進血管新生,進而促進子宮內膜異位組織病灶的生長。此研究證實了雙特異性去磷酸酶-2在子宮內膜異位症的發展過程中調節了缺氧所誘導的促血管生成因子,並提出介白素-8可能成為一個可能的治療標的。
英文摘要 Endometriosis is one of the most common gynecological disorders, defined as the presence of endometrial glands and stroma outside of the uterine cavity. The pathogenic mechanism of endometriosis is complicated and is largely unknown. It has been well-accepted that endometriotic lesions originated from refluxed endometrial tissues, which have to gain the ability of neovasculization to survive under the hypoxic environment of ectopic sites. Our previous study has shown that dual specificity phosphatase-2 (DUSP2), a mitogen activated protein kinase (MAPK)-specific nuclear phosphatase, is downregulated in endometriotic tissues in a hypoxia inducible factor-dependent manner and contributes to the pathological process of endometriosis. Although hypoxia plays important roles in angiogenesis, whether DUSP2 is involved in angiogenesis during the development of endometriosis remains unknown. This study was designed to investigate the biological functions of hypoxia-mediated DUSP2 downregulation on angiogenesis during the development of endometriosis. Whole genome screening data revealed that DUSP2 regulated a subset of angiogenic genes, we chose interleukin-8 (IL-8) for subsequent characterization since it is a potent angiogenic factor. Quantitative RT-PCR results demonstrated that the levels of IL-8 were elevated in ectopic endometriotic stromal cells, which was inversely correlated with levels of DUSP2. Results also showed the induction of IL-8 could be recapitulated by hypoxia treatment or DUSP2 knockdown in eutopic endometrial stromal cells. Promoter activity assay results showed that hypoxia-mediated DUSP2 downregulation enhanced IL-8 expression through a transcriptional regulation by CCAAT/enhancer binding protein α (C/EBPα). Hypoxia-induced binding of C/EBPα to IL-8 promoter was evidenced by chromatin immunoprecipitation-PCR assay. Treatment with conditioned medium collected from endometrial stromal cells cultured under hypoxia or with DUSP2 knockdown promoted network formation of human umbilical vein endothelial cells, which can be blocked by administration of IL-8 receptor inhibitor. Finally, results from in vivo angiogenic mouse model demonstrated the number of autoimplant-induced endometriotic-like lesions and the microvasculature within the lesions were significantly reduced by blocking the signaling of IL-8. Taken together, results from this study demonstrate that loss-of-DUSP2 in endometriotic stromal cells causes IL-8 upregulation, which results in increased angiogenic capacity and promotes the growth of endometriotic lesions. This study provides solid evidence to demonstrate that hypoxia-regulated DUSP2 downregulation promotes expression of angiogenic factor, IL-8, during the development of endometriosis and suggests it may serve as a potential therapeutic target.
論文目次 Certificate of Examination I
Abstract in Chinese I
Abstract in English IV
Acknowledgement VI
Table of Contents VIII
List of Tables X
List of Figures XI
Introduction 1
Endometriosis 1
Etiology 1
Inflammation and endometriosis 2
Hypoxia in endometriosis 3
Angiogenesis 7
Interleukin-8 7
Interleukin-8 and endometriosis 10
Hypothesis 10
Materials and Methods 11
Patients and study population 11
Isolation of stromal cells 11
Cell culture and treatment 12
RNA isolation 12
Reverse transcription and quantitative realtime PCR (RT-qPCR) 13
RNA interference knockdown 14
Plasmid, transfection and promoter activity assay 14
Chromatin immunoprecipitation (ChIP) assay 15
In vitro tube formation assay 16
Animals 17
Surgical induction of endometriosis in mouse model 17
Histology and immunohistochemistry 18
Statistical analysis 18
Results 22
Expression of IL-8 is up-regulated by hypoxia treatment and DUSP2 knockdown 22
Hypoxia and DUSP2 knockdown transcriptionally upregulate IL-8 expression via ERK activation 25
CCAAT enhancer binding site is critical for hypoxia and DUSP2 knockdown-induced IL-8 expression 25
Hypoxia and loss-of-DUSP2-induced IL-8 promotes angiogenesis in vitro 31
IL-8 promotes the formation of endometriosis in vivo 31
Discussion 38
Reference 43
Appendix 51
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