臺灣博碩士論文加值系統

肺部上皮細胞在肺纖維化疾病中扮演重要角色。本研究將探討在人類肺部上皮細胞(A549)中，TGF-β誘導的CTGF表現及EMT作用是否受到extracellular signal-regulated kinase (ERK)/ADAM17/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ)訊號路徑的調控。結果顯示，TGF-β能夠大量誘導CTGF表現，且受到ADAM17 small interfering RNA (siRNA)、RSK1 siRNA、C/EBPβ siRNA、TAPI-0 (ADAM17抑制劑)和U0126 (ERK抑制劑)抑制。TGF-β可以增強ERK磷酸化，且TGF-β促進的ADAM17磷酸化受到U0126抑制。再者，TGF-β刺激的RSK1磷酸化受到TAPI-0和U0126的抑制。TGF-β可以刺激C/EBPβ磷酸化，並被U0126、ADAM17和RSK1 siRNA抑制。TGF-β也能夠促使C/EBPβ結合於CTGF promoter。此外，TGF-β可以增強fibronectin (FN)、collagen I和CTGF mRNA表現，而降低E-cadherin mRNA的表現。最後，TGF-β也誘導了FN蛋白的產生，且受到TAPI-0、ADAM17 siRNA及CTGF siRNA的抑制。綜合以上結果，我們發現在人類肺部上皮細胞中，TGF-β經由活化ERK/ADAM17/RSK1/C/EBPβ訊息傳遞路徑，誘導CTGF的表現進而媒介EMT的作用。

Lung epithelial cells play important roles in pulmonary fibrosis. In this study, we investigated whether TGF-β-induced CTGF expression and EMT formation was regulated in extracellular signal-regulated kinase (ERK)/ADAM17/ribosomal S6 kinases 1 (RSK1)/ CCAAT/enhancer-binding protein β(C/EBPβ) signaling pathway in human lung epithelial cells (A549). We found that TGF-β induced CTGF expression weakened by ADMA17 small interfering RNA (ADAM17 siRNA), RSK1 siRNA, C/EBPβ siRNA, TAPI-0 (an ADAM17 inhibitor) and U0126 (an ERK inhibitor). TGF-β induced ERK phosphorylation as well as ADAM17 phosphorylation, which was attenuated by U0126. TGF-β caused increase in RSK1 phosphorylation inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. Also, TGF-β increased the recruitment of C/EBPβ to CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), collagen I, and CTGF mRNA levels as well as reduced E-cadherin mRNA level. Moreover, TGF-β-stimulated FN protein expression was attenuated by TAPI-0, and ADAM17 and CTGF siRNA. Taken together, these results suggested that TGF-β activates ERK/ADAM17/RSK1/C/EBPβ signaling pathway, which in turn induces CTGF expression and ultimately mediates EMT process in human lung epithelial cells.

壹、中文摘要......1
貳、英文摘要......3
参、緒論.........5
1. 肺部纖維化疾病......6
2. 轉化生長因子-β......7
3. 上皮-間質細胞轉換......8
4. A disintegrin and metalloproteinase 17 (ADAM17)...9
5. 結締組織生長因子......11
6. ERK/RSK1/C/EBPβ對CTGF的活化調控機制......12
肆、實驗材料與方法......14
一、實驗材料......15
二、方法......16
1. 細胞培養......16
2. siRNAs 轉染......16
3. 西方墨點法......17
4. 染色質免疫沈澱分析法......18
5. 定量逆轉錄聚合酶鏈式反應......19
6. 統計方法......20
伍、結果......21
1. TGF-β誘導人類肺部上皮細胞CTGF的表現......22
2. ADAM17活化參與TGF-β誘導之CTGF表現......22
3. ERK媒介TGF-β誘導之CTGF表現及ADAM17活化......23
4. RSK1媒介TGF-β誘導之CTGF表現......24
5. C/EBPβ媒介TGF-β誘導之CTGF......25
6. TGF-β經由ERK、RSK1、ADAM17活化C/EBPβ......25
7. TGF-β誘導之EMT作用......26
8. ADAM17及CTGF媒介TGF-β誘導之FN表現......26
陸、討論......28
未來展望......34
柒、參考文獻......35
捌、圖表......44

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