Peroxisome proliferator-activated receptor （PPAR）γ coactivator-1α （PGC-1α）是一種轉錄活化因子，參與能量代謝和粒腺體生合成。在糖尿病病患中代謝性疾病和腎纖維化、末期腎病息息相關。在糖尿病大鼠中PGC-1α的活化表現影響肝臟和胰臟β細胞的功能。在本篇研究中我們試著探討PGC-1α在TGF-β1所誘導的腎間質纖維化細胞模型中所扮演的角色。方法：細胞培養腎纖維母細胞（NRK-49F）並利用TGF-β1 （5 ng/ml）24小時刺激誘導，以及轉染pcDNA3.1（1.5 μg/well）、pcDNA3.1-PGC-1α （1.5 μg/well）或PGC-1α-siRNA（10 μM），運用西方墨點試驗和免疫螢光分析PGC-1α、纖維蛋白、第一型膠原蛋白的表現。結果：我們發現在加入TGF-β1後細胞增加纖維蛋白及第一型膠原蛋白的表現，並同時誘導PGC-1α增加（**P<0.01）。在轉染試驗中也發現PGC-1α的表現量增加會誘導纖維蛋白及第一型膠原蛋白的表現，然而在西方墨點法和免疫螢光法中發現轉染PGC-1α-siRNA則會逆轉TGF-β1所誘導纖維蛋白及第一型膠原蛋白的表現（**P<0.01）。此外，在加入5 μM 的 SB431542　（TGFβ-RI）、LY294002 （PI3K/Akt） 和SB203580 （p38 MAPK）的訊息抑制劑後會抑制TGF-β1所誘導增加的PGC-1α。結論：綜合以上結果，我們認為在NRK-49F細胞株中PGC-1α誘導纖維蛋白及第一型膠原蛋白增加是透過TGF-β1誘導 TGFβ-RI、PI3K/Akt和p38 MAPK訊息路徑。這些數據顯示，在腎間質纖維化中PGC-1α 是一個潛在的治療標的。

Peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α) is a transcriptional coactivator that regulates energy metabolism and mitochondrial biogenesis. Metabolic syndrome is associated with a higher prevalence of renal fibrosis and end stage renal disease (ESRD) in diabetic patients. PGC-1α expression and activity was increased in the liver and pancreatic β cells in animal models of diabetes mellitus. The aim of present study was to investigate the fibrosis regulatory role of PGC-1α. Method: Cultured NRK-49F cells were treated with TGF-β1 (5 ng/ml) for 24 h to establish an in vitro renal fibrosis model. Cells were transfected with pcDNA3.1 (empty vector, 1.5 μg/well), pcDNA3.1-PGC-1α (1.5 μg/well), or PGC-1α-siRNA (10 μM). Western blotting and Immunofluorescence were used to evaluate the expression of fibronection, type I collagen and PGC-1α Results: TGF-β1-induced an increase in fibronection, type I collagen and PGC-1α by Western blotting assays (##P<0.01). In addition, treatment of pcDNA3.1-PGC-1α significantly induced an increase in fibronectin and type I collagen (#P<0.05). Treatment of PGC-1α-siRNA significantly reversed TGF-β1-induced increases in the level of fibronectin and type I collagen compared to control-siRNA (**P<0.01) according to Western blotting and immunofluorescence analysis. Moreover, 5 μM of SB431542, LY294002 or SB203580, the pathway inhibitors of TGFβ-RI, PI3K/Akt, p38 MAPK respectively, suppressed the TGF-β1-induced increase in the level of PGC-1α (**P<0.01). Conclusion: We suggest that PGC-1α has the potential to regulated renal interstitial fibrosis through TGF-β1-induced TGFβRI, PI3K/Akt and p38 MAPK signaling pathways. Therefore PGC-1α may be used as a potential therapeutic target for treating renal fibrosis.

口試委員審定書 i
致謝 v
中文摘要 vi
英文摘要 viii
縮寫表 x
第一章 緒論 1
一、 腎纖維化之影響 2
二、 腎纖維化之機轉 2
三、 纖維化的生物指標 3
四、 乙型轉化生長因子 4
五、 乙型轉化生長因子的活化路徑和纖維化的調節 5
六、 Peroxisome proliferator-activated receptor (PPAR)γ coactivator-1α (PGC-1α) 6
七、 PGC-1α的調節與疾病 7
第二章 研究目的 9
第三章 材料方法 11
一、 材料 12
二、 細胞培養 12
1. 解凍細胞 13
2. 繼代培養 13
3. 冷凍保存 13
4. 訊息傳遞路徑抑制劑 14
三、 細胞檢體製備 14
四、 質體的製備與抽取 15
五、 質體轉染 16
六、 siRNA轉染 16
七、 西方墨點法(Western blotting) 17
八、 免疫螢光法(Immunoflurescence) 18
九、 統計分析 19
第四章 結果 20
一、 TGF-β1誘導纖維蛋白、第一型膠原蛋白、TGFβ-RI、TGFβ-RII、Smads及PGC-1α的影響 21
二、 TGF-β1在不同時間點誘導PGC-1α的影響 21
三、 轉染pcDNA3.1-PGC-1α質體誘導PGC-1α、纖維蛋白和第一型膠原蛋白的影響 21
四、 轉染PGC-1α-siRNA逆轉TGF-β1誘導PGC-1α、纖維蛋白和第一型膠原蛋白的影響 22
五、 SB431542、LY294002、SB203580訊息抑制劑對TGF-β1誘導PGC-1α的影響 23
第五章 結論與討論 24
第六章 圖表 28
第七章 參考文獻 35

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