臺灣博碩士論文加值系統

許多器官及組織會發生纖維化的現象，以腎臟而言，纖維化則是慢性腎臟疾病 (chronic kidney disease) 常見且重要的特徵。而α-平滑肌肌動蛋白 (α-SMA) 常被當作是纖維化的生物標記，已知乙型轉化生長因子 (TGF-β) 會促進α-SMA的生成，並且增加Smad蛋白質的磷酸化，進而去影響上皮-間質轉化及細胞週期。文獻指出由成纖維細胞分泌的五甲氧基色胺酸 (5-Methoxytryptophan, 5-MTP) 具有減少上皮-間質轉化及消炎的效果，但對由TGF-β所誘發的纖維化影響尚未清楚。葡萄糖胺鹽酸鹽 (glucosamine hydrochloride, Gln) 則有研究指出可以減弱TGF-β訊號的傳遞，進而改善由其所誘發的腎臟纖維化。因此本論文主要以 5-MTP 對照Gln利用腎臟成纖維細胞NRK-49F細胞株來探討對TGF-β所引發的腎臟纖維化是否有抑制效果及其作用的可能機轉。從結果觀察到TGF-β誘導纖維化現象的最佳處理時間與劑量分別是24小時及5ng/ml，也發現細胞死亡與細胞週期改變與作用時間較有關連。接著觀察到不論是加入5-MTP或是Gln都可以減少TGF-β所造成的蛋白質表現量及細胞週期之影響。另外以四種不同信息路徑抑制劑觀察TGF-β的作用，發現JNK、PI3K及Akt/p38抑制劑會抑制TGF-β對α-SMA蛋白量的增加，並增加Smad2磷酸化的量；MAPKK抑制劑則反之。另外也發現JNK及PI3K抑制劑會加強5-MTP對TGF-β對α-SMA蛋白量的抑制效果，同時增加Smad2磷酸化的量；JNK、PI3K及MAPKK會加強Gln對TGF-β對α-SMA蛋白量的抑制效果，且能完全抑制Smad2的磷酸化。利用核質分離技術主要探討PI3K路徑，可以發現在核中的p65在Gln加入下有減少。綜合目前的結果可得知5-MTP如同Gln對於TGF-β所造成的腎臟纖維化具有減緩的能力，然而兩者對蛋白質以及基因的影響卻不一致。在機轉部分僅可得知 5-MTP 與Gln 可能是經由不同傳遞路徑來達到抑制效果，5-MTP 可能是透過非Smad依賴性的路徑；而 Gln 可能是透過 Smad 依賴性路徑。以及 TGF-β 造成腎臟纖維化的機轉可能是Smad依賴性與非Smad依賴性之傳遞路徑同時進行，詳細的機轉尚需更多實驗來進行驗證。

Fibrosis occurs in many organs and tissues and it is a common and important feature of chronic kidney disease in kidney. α-smooth muscle actin (α-SMA) is generally regarded as a biomarker of fibrosis, which is induced by several agents, including transforming growth factor beta (TGF-β). phosphorylation of Smad proteins by TGF-β is to affect Epithelial-to-mesenchymal transition and cell cycle. Previous studies have indicated that 5-methoxytryptophan (5-MTP) secreted by fibroblasts has the effect of reducing EMT and anti-inflammatory, but it is unclear whether works on the effect of fibrosis induced by TGF-β. Glucosamine hydrochloride (Gln) has been shown to ameliorate renal fibrosis induced by TGF-β. In this study, we mainly compared 5-MTP with Gln whether suppressed TGF-β-induced fibrosis in the renal fibroblast cell line, NRK-49F. First, TGF-β induced fibrosis and the optimal promotion effect was obtained according to the treatment time and dose of 24 hours and 5 ng/ml, respectively. Also, cell death and change of cell cycle were time dependent. Then, the addition of 5-MTP or Gln both reduced the levels of TGF-β-induced protein expression and change of cell cycle. In addition, four different signal pathway inhibitors were used to examine the potential regulation of TGF-β effects. Our results showed that inhibitors of JNK, PI3K and Akt/p38 inhibited the induction of α-SMA protein by TGF-β. Instead of Akt/P38 increased the phosphorylation of Smad2, MAPKK increased the amount of α-SMA protein and decreased the phosphorylation of Smad2. Furthermore, JNK and PI3K inhibitor enhanced the inhibitory effect of 5-MTP on the induction of α-SMA protein by TGF-β and increased the phosphorylation of SMAD2. JNK, PI3K and MAPKK inhibitor enhanced the inhibitory effect of Gln on the TGF-β-induced α-SMA protein, but completely inhibited the phosphorylation of Smad2. Then, using the nuclear fraction technology to mainly explore the PI3K pathway, it could be found that p65 in the nucleus was reduced by Gln addition. Summarily, 5-MTP had the ability to suppress renal fibrosis induced by TGF-β, but the effects of 5-MTP and Gln on the protein and gene were inconsistent. And we can know in mechanism only 5-MTP and Gln via different pathways to achieve inhibition. 5-MTP may be through non-Smad pathway and Gln may be through Smad pathway. And the mechanism of TGF-β inducing renal fibrosis may be through Smad and non-Smad pathways at same time. It still needs more experiments to confirm detailed mechanism.

目錄
目錄 I
圖目錄 III
表目錄 IV
縮寫表 V
中文摘要 VI
英文摘要 VIII
第一章 緒論 1
第一節 纖維化 1
第二節 乙型轉化生長因子 (TGF-β) 1
第三節 NRK-49F cell line 2
第四節 Smad依賴性與非Smad依賴性途徑 2
第五節 5-甲氧基色胺酸 (5-MTP) 3
第六節 細胞週期 3
第七節 研究目標與動機 4
第二章 材料與方法 5
第一節 實驗材料 5
第二節 實驗方法 9
第三章 結果 19
第一節 TGF-β 作用不同時間與濃度之影響 19
第二節 細胞培養液的生長因子對於 TGF-β 所造成影響之關係 20
第三節 不同濃度的 5-MTP 對於 TGF-β 效果之影響 21
第四節 5-MTP、Gln與 TGF-β 加入的先後順序是否有影響 22
第五節 非 Smad 依賴性的四種路徑抑制劑對 TGF-β 效果之影響 22
第六節 四種路徑抑制劑對 5-MTP、Gln 抑制 TGF-β 效果之影響 23
第七節 TGF-β 對 Smad 依賴性路徑以及 PI3K 路徑訊號之影響 23
第八節 TGF-β對其他細胞之影響 25
第四章 討論 27
第一節 TGF-β造成 NRK-49F 細胞死亡的原因 27
第二節 探討 TGF-β 造成纖維化的可能訊號傳遞路徑 28
第三節 基因與蛋白質表現不同步 29
第五章 結論 31
第六章 參考資料 51

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