肝臟受到如病毒性肝炎、酒精造成之肝病等傷害，會促使肝臟星狀細胞(hepatic stellate cells)的增生與活化，並誘發一連串的組織修補及重塑(remodeling)機制。其中包括肝內細胞激素分泌，尤其是 TGF-β1(transforming growth factor-β1)的生成，是直接刺激肝臟星狀細胞分泌胞外基質(extracellular matrix)，加重肝纖維化的主要細胞激素之一。
而Bone Morphogenetic protein-2 (BMP-2)蛋白隸屬TGF-β superfamily成員之一，已知具有調控細胞生長、分化及骨質生成等功能。先前的動物實驗數據指出，肝內BMP-2 蛋白含量變化會隨著肝臟纖維化過程中TGF-β1 含量增加而遞減。BMP-2 的減少有可能會失去擷抗TGF-β1 所引起的肝臟纖維化的能力。而本研究目的即在證實BMP-2與TGF-β1 二者間在傳遞訊息路徑中是否存在具有相互擷抗性的調控機制。
本實驗利用正常肝細胞Clone-9 cell 與肝臟星狀細胞HSC-T6 cell兩株細胞中加入外生性重組蛋白TGF-β1，實驗發現TGF-β1 隨著不同劑量的增加相對的BMP-2 表現量也有隨之減少。相反之，BMP-2隨著不同劑量的增加相對的TGF-β1 也會逐漸下降。實驗數據證實在兩者間，不但在動物體內實驗模式有相互調節作用的現象，在體外細胞實驗也會有相同結果。利用西方墨點法分析TGF-β1 與BMP-2 的訊息傳遞路徑，顯現出不同的蛋白激酶的磷酸化現象，但是TGF-β1與BMP-2 在引起纖維化過程中，交互作用的訊息傳遞路徑與更詳細的機制仍需未來更進一步的研究。

Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-β1 (TGF-β1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-β1 (TGF-β1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-β1 superfamily, has been known to regulate cell proliferation, differentiation, and bone morphogenesis. Our previous study demonstrated that BMP-2 was downregulated in fibrotic liver of mice, supporting its antagonistic role in hepatic fibrogenesis. Downregulation of BMP-2 in fibrotic livers may lose its ability to antagonize the pro-fibrogenic action of TGF-β1. The purpose of this study was to determine whether mutual regulatory mechanisms exist between BMP-2 and TGF-β1.
Treatment of recombinant protein, TGF-β1, significantly suppressed BMP-2 expression in hepatocytes clone-9 and HSC-T6 cells. Moreover, treatment of BMP-2 in both cell also attenuated TGF-β1protein levels in a dose-dependent manner. This finding supports that TGF-β1 and BMP-2 mutually modulated their expression not only in vivo, but also in vitro. Western blotting analysis showed that TGF-β1 and BMP-2 exerted different kinase phosphorylation profiles of signaling activities. However, the signal pathways and detail mechanisms of the interactions of BMP-2 and TGF-β1 in the fibrogenic action will need to further evaluate.

目錄 …………………………………………… 5
縮寫 …………………………………………… 6
中文摘要 ……………………………………… 7
英文摘要 ……………………………………… 9
前言 …………………………………………… 11
實驗背景 ……………………………………… 17
實驗目的 ……………………………………… 18
實驗方法與材料……………………………… 19
結果 …………………………………………… 28
討論 …………………………………………… 32
圖表…………………………………………… 35
附圖…………………………………………… 45
參考文獻 ……………………………………… 49

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