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研究生:鄭凱元
研究生(外文):Kai-Yuan Jheng
論文名稱:環孢素在牙齦上皮細胞中經由轉化生長因子β及離氨基氧化酶樣蛋白2 誘導上皮-間質轉換
論文名稱(外文):Cyclosporin-A induced EMT through TGF-β and LOXL-2 pathways in gingival epithelial cells
指導教授:郭彥彬郭彥彬引用關係
口試委員:周涵怡張瑞青
口試日期:2019-07-31
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:32
中文關鍵詞:環孢素牙齦過度增生轉化生長因子-β上皮-間質細胞轉換離氨基氧化酶樣蛋白2
DOI:10.6342/NTU201903783
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實驗目的和背景:
環孢素(Cyclosporin A)是一種常用在器官移植患者上的免疫抑制藥物,大約有七成服用此藥的病人會產生牙齦過度增生(Gingival overgrowth,GO)的副作用,進而影響到說話、吞嚥、咀嚼、口腔衛生、美觀等等,影響生活品質。轉化生長因子-β(Transforming growth factor,TGF-β) 在牙齦腫大的致病機轉中扮演著主要的角色,先前的研究中發現環孢菌素可經由TGF-β傳導路徑誘發上皮-間質轉換(epithelial-mesenchymal transition,EMT)現象,而使患者牙齦增生。其中離氨基氧化酶樣蛋白2 (Lysyl Oxidase-like protein 2, LOXL-2):除催化膠原蛋白與彈性纖維的交叉共價鍵結(cross-linking)外,在一些研究中指出和EMT現象有關連。本研究期望能更加了解環孢素是否經由LOXL-2誘導人類牙齦上皮細胞產生EMT。
實驗方法:
本研究利用西方墨點法來檢測環孢素-A以及不同抑制劑處理後人類牙齦上皮細胞OECM-1 及 Ca9-22 之LOXL-2的表現以及EMT 標識蛋白Slug及E-cadherin的變化。
實驗結果:
於 OECM-1 及 Ca9-22 細胞加入環孢素素處理後,其 LOXL-2 的表現量隨之增加,OECM-1 細胞在4小時後達到最高值;CA9-22 細胞是在6小時後達到最高值。前處理TGF-β 中和抗體 (20μg/ml)、ALK5 抑制劑SB431542 (20μg/ml)、Smad-3 抑制劑SIS3 (5 μg/ml),可以抑制OECM-1及CA9-22細胞受環孢素誘導的LOXL-2表現。顯示環孢素是經由TGF-β訊息傳導路徑誘導 OECM-1 及 CA9-22 之LOXL-2 的表現。前處理LOXL-2 的抑制劑 BAPN,可以顯著的抑制環孢素誘導的OECM-1 及 Ca9-22細胞上皮-間質轉換標識蛋白Slug的表現增加及E-cadherin的減少。顯示LOXL-2在Cyclosporin A誘導的EMT扮演重要的角色。
結論:
環孢素經由TGF-β及LOXL-2誘導EMT的產生。
Objectives:
Cyclosporin A (CsA) is an immunosuppressive drug commonly used in organ transplant patients. Approximately 70% of the patients show gingival overgrowth (GO). Transforming growth factor (TGF-β) plays a major role in the GO. Previous studies have found that Cyclosporin A can induce epithelial-mesenchymal transition (EMT) via TGF-β signaling pathway, which then plays important roles in the pathogenesis of GO. Lysyl oxidase-like protein 2 (LOXL-2) : catalyzes the cross-linking of collagen and elastin to maintain the rigidity and stability of the extracellular matrix protein. Previous studies have shown LOXL-2 plays important roles in EMT. This study investigated the signaling pathways involved in the CsA-induced EMT.

Material and Methods:
Western blotting was used to examine the levels of LOXL-2 protein and the EMT marker protein E-cadherin and Slug in human gingival epithelial OECM-1 and Ca9-22 cells after treatment with CsA and various inhibitors.

Results:
CsA induced LOXL-2 in OECM-1 and Ca9-22 cells in a dose- and time- dependent manner. Pretreatment with TGF-β neutralizing antibody, ALK5 inhibitor SB431542 and Smad-3 inhibitor SIS3 almost completely inhibit cyclosporin A-induced LOXL-2 expression in OECM-1 and CA9-22 cells. These results suggest that CsA-induced LOXL-2 expression in gingival epithelial cells is mediated through TGFβ1 signaling. Pretreatment with LOXL-2 inhibitor BAPN significantly reduced CsA-induced Slug protein levels and reversed CsA-reduced E-cadherin expression in OECM-1 and CA9-22 cells.

Conclusion:
Cyclosporin A induced EMT through TGFβ signaling and LOXL-2 protein expression.
謝誌 i
中文摘要 ii
Abstract iii
目錄 iv

導論 1
第一節 牙齦過度增生 1
1-1 牙齦過度增生 1
1-2 牙齦過度增生的流行病學 2
1-3 牙齦過度增生的致病機制 3
1-4 牙齦過度增生的治療 3
第二節 環孢素(Cyclosporin A, CsA) 3
2-1 Cyclosporin A 的簡介 4
2-2 Cyclosporin A 的作用機轉 4
2-3 Cyclosporin A 與牙齦過度增生 4
第三節 轉化生長因子-β1(TGF-β1) 5
3-1 TGF-β1 的簡介 5
3-2 TGF-β1的訊息傳遞路徑 6
3-3 TGF-β1 與纖維化 7
第四節 離氨基氧化酶樣蛋白-2 (LOXL-2) 9
4-1 LOXL-2 的簡介 9
4-2 LOXL-2 與TGF-β1的訊息傳遞路徑及纖維化 9
研究目的 10
材料與方法 11
第一節 細胞株與細胞培養 11
第二節 藥物處理 11
2-1 Cyclosporin A藥物處理 11
2-2 抑制劑、中和抗體使用資料 11
第三節 西方墨點法 12
3-1 蛋白萃取 12
3-2 膠體配置與電泳分析 12
3-3 蛋白轉漬 12
3-4 抗體反應與顯影呈色 13
第四節 統計與方法 13
結果 14
Cyclosporin A可誘導牙齦上皮細胞OECM-1及CA9-22之LOXL-2的表現 14
Cyclosporin A經由TGF-β訊息傳導路徑誘導OECM-1及CA9-22之LOXL-2表現 14
LOXL-2在Cyclosporin A誘導的EMT扮演重要的角色 14
討論 15
圖與表 17
參考文獻 23
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