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研究生:陳韋銘
研究生(外文):Chen, Wei-Ming
論文名稱:探討20-Hydroxyecdysone於第一型乙型轉型生長因子誘導腎近端小管細胞纖維化的角色
論文名稱(外文):The role of 20-hydroxyecdysone in attenuating TGF beta-1-induced renal cellular fibrosis in proximal tubule cells
指導教授:楊堉麟楊堉麟引用關係
指導教授(外文):Yang, Yu-Lin
口試委員:莊麗月顧進裕
口試委員(外文):Chuang, Lea-YeaGuh, Jinn-Yuh
口試日期:2011/06/23
學位類別:碩士
校院名稱:中華醫事科技大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:72
中文關鍵詞:腎纖維化乙型轉型生長因子上皮-間質轉化20-去氫蛻皮激素
外文關鍵詞:Renal fibrosisTGF-betaEpithelial-to-Mesenchymal transition (EMT)20-hydroxyecdysterone (20-HE)
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纖維化是末期腎病變的主要特徵,造成腎小管不可逆性的損傷。先前的文獻指出,在糖尿病動物模型20-hydroxyecdysone (又稱20HE) 能夠降低腎臟損傷。然而20HE對於腎臟纖維化的調控角色仍然需要進一步的研究。方法: 細胞培養近端腎小管上皮細胞 (HK-2) 利用48小時乙型轉型生長因子TGF-1的刺激誘導作為腎細胞纖維化的體外模型,透過不同濃度的20HE (0到50M) 處理24小時,以觀察細胞內外的蛋白質的改變。利用酵素連結免疫吸附法分析細胞外的纖維蛋白表現。西方墨點法與免疫螢光染色偵測乙型轉型因子訊息路徑(TGF-1/Smad pathway) 的訊息傳遞蛋白(包含Smad2/3,4以及7),上皮-間質轉換的標記蛋白 (e.q. E-cadhirin與 -smooth muscle actin) 以及EMT的轉錄調節者Snail蛋白的表現。結果: 20HE逆轉TGF-1所造成細胞內外纖維蛋白的堆積。同時20HE能夠增加TGF-1的負向調節蛋白Smad 7的表現,降低Smad 2/3 的蛋白質表現以及磷酸化。另一方面20HE同時抑制上皮-間質的轉換過程。並且也抑制Snail 蛋白的表現。 結論:我們的研究指出20HE 在近端腎小管上皮細胞的纖維化模型中具有對抗纖維形成的潛力,更重要的是20HE能夠對抗纖維形是透過調節乙型轉型生長因子Smad訊息路徑和上皮-間質轉換過程。
Renal fibrosis is end stage of diabetes kidney diseases, which causes irreversible progressive proximal tubular injury. In the previous study, 20-hydroxyecdysterone (20HE) attenuated renal injury in diabetes models. However, the fibrosis regulatory role of 20HE remains to be investigated. Methods: The proximal tubular epithelial cell (designated as HK-2) were treated for 48 hours with TGF-1 (5 ng/ml) in different concentrations of 20HE (0 to 50 M/ml) in the last 24 hours of culture. The extracellular fibronectin was measured by ELISA assay. Western blot and immunofluorescence were used to evaluate the expression of TGF-1/Smads transducer (including Smad2/3, 4, and 7), epithelial and mesenchymal markers (e.q. E-cadhirin and α-smooth muscle actin) and snail (a transcriptional regulator for EMT). Results: 20HE reversed TGF-1-induced increase in fibronectin (both intracellular and extracellular fibronectin). Simultaneously, 20HE reversed TGF-1-induced down- regulation of Smad7. In addition, 20HE attenuated TGF-1-induced up-regulation of Smad 2/3 and its phosphorylation. 20HE was significantly restored in TGF--induced downregulation of E-cadherin and prohibited gained expression of -SMA. In addition, snail proteins were down-regulated by 20HE. Coclusion: We suggest that 20HE is a potential fibrosis antagonist for proximal tubule cells. More importance, 20HE appear to be an anti-fibrosis agent was through regulation of TGF-1/Smad pathway and EMT.
總目錄
致謝 iii
中文摘要 v
英文摘要 vi
縮寫表 viii
作者簡歷 ix
第一章 緒論 1
一、慢性腎臟病之盛行率 2
二、腎臟纖維化之致病與機轉 3
三、腎臟纖維化與EMT過程之相關 5
四、轉型生長因子與慢性腎臟病之關係 7
五、纖維化之訊息傳導路徑 8
六、20-hydroxyecdysone簡介 11
第二章 研究目的 16
第三章 材料與方法 18
1. 細胞培養 19
( 1 ) 繼代細胞 19
( 2 ) 活化細胞 20
( 3 ) 細胞保存 21
2. 酵素連結免疫吸附分析( ELISA ) 21
3. 細胞Immunofluorescence分析 22
4. 細胞蛋白質總量測定 23
5. 西方墨點分析 24
第四章 結果 27
一、20-去氫蛻皮激素逆轉乙型轉型生長因子所造成人類腎小管上皮細胞型態改變 28
二、20-去氫蛻皮激素減少在人類腎小管上皮細胞乙型轉型生長因子所誘導的纖維蛋白增加 28
三、20-去氫蛻皮激素透過抑制訊息傳遞抑制乙型轉型生長因子所誘導的纖維化 29
四、20-去氫蛻皮激素恢復乙型轉型生長因子所誘導的上皮間質轉換 30
五、20-去氫蛻皮激素透過調節Snail抑制乙型轉型生長因子所誘導的上皮間質轉換 31
第五章 結論與討論 32
第六章 圖表 37
第七章 參考文獻 47

圖表目錄
圖1. 20HE逆轉TGF-β1在腎小管上皮細胞所誘導的細胞型態改變。 38

圖 2. 20HE降低TGF-b1所誘導近端腎小管上皮細胞,細胞內纖維蛋白的表現。 39

圖 3. 20HE降低TGF-b1所誘導近端腎小管上皮細胞,細胞外纖維蛋白的堆積。 40

圖 4. 20HE降低TGF-b1所誘導近端腎小管上皮細胞,細胞內TRI和TRII的表現。 41

圖 5. 20HE降低TGF-b1所誘導近端腎小管上皮細胞,細胞內Smad 2/3, pSmad 2/3和Smad 7的表現。 42

圖 6. 20HE降低TGF-b1所誘導近端腎小管上皮細胞,細胞內E-cadherin和-SMA的表現。 43

圖 7. 20HE經由調控EMT的機制抑制TGF-β1誘導細胞纖維化的機轉。 44

圖 8. 20HE降低TGF-b1所誘導近端腎小管上皮細胞Snail 蛋白的表現。 45

圖 9. 20HE抑制TGF-β1所誘導的纖維形成的機制。 46

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