樺木酸（Betulinic acid；BetA）是由白樺樹皮所萃取出的三萜類化學物質，被認為對癌細胞與腫瘤細胞具有毒性。而樺木酸對細胞中特殊受體的影響目前尚無廣泛且清楚的研究，故針對樺木酸對轉化生長因子β（TGF-β）訊息傳遞的影響進行探討。TGF-β是調節細胞增殖與細胞遷移的關鍵調節物，同時也參與癌症的發生與發展進程。TGF-β藉由Smad蛋白之依賴性與非依賴性的訊息傳遞路徑，來調節腫瘤細胞的增殖和侵襲。實驗中藉由蔗糖濃度梯度超高速離心法及西方墨點法確認：在大多數細胞中，TGF-β受體主要位於脂筏/caveolae微區。而樺木酸在不改變TGF-β受體總量的情況下，能夠誘導TGF-β受體從脂筏/caveolae 移動至non-caveolae微域。目前已發現TGF-β受體於脂筏/caveolae與clathrin所調控的內吞作用之間的分布情形，可調節TGF-β的反應程度。最值得注意的是，樺木酸所誘導的TGF-β受體易位是非常快速的，同時也與TGF-β所誘導的訊號相關。於是我們提出樺木酸可將TGF-β受體從脂筏/caveolae移出，以增強TGF-β訊息傳遞並且顯著增加TGF-β刺激Smad2磷酸化的能力。此外，在水貂肺部上皮細胞（Mv1Lu cell）中，樺木酸所引起TGF-β受體重新分布的情形，可以增強TGF-β所誘導的PAI-1報導基因的活性，並增強細胞生長抑制。這些結果顯示，樺木酸的作用可能是源於細胞膜上一部份TGF-β受體在non-caveolae微域中增強了訊息強度所致。

Betulinic acid (BetA) is a phytochemical triterpenoid acid from bark extracts and is cytotoxic to cancer cells and tumors. The specific subcellular receptors that are bound or deactivated by BetA have not been extensively investigated or identified. The goal of this study is to investigate the effect of BetA in transforming growth factor β (TGF-β) signaling. TGF-β is a key modulator in regulating cell proliferation and migration, it also involved in the process of cancer development and progression. TGF-β regulates tumor cell proliferation and invasion through a variety of Smad-dependent and -independent pathways. In most of cells, TGF-β receptors were located predominantly in lipid raft/caveolae microdomains, determined using sucrose density gradient ultracentrifugation and Western blot. BetA induces translocation of TGF-β receptors from lipid raft/caveolae to non-caveolae microdomains without changing total level of TGF-β receptors. Distribution of TGF-β receptors between lipid raft/caveolae- and clathrin-mediated endocytosis has been found to regulate TGF-β responsiveness. Most notably, BetA-induced TGF-β receptors translocation is rapid and correlate with the TGF-β-induced signaling. Here, we provide for the first the time evidence that BetA enhanced TGF-β signaling is associated with translocation of TGF-β receptors out of lipid raft/caveolae microdomains and a dramatic increase in the ability of TGF-β to stimulate Smad2 phosphorylation. Moreover, BetA-induced redistribution of TGF-β receptors also enhances TGF-β -induced PAI-1 reporter gene activation and growth inhibition in Mv1Lu cells. These results implicated that anticancer activities of BetA may be due, in part to the enhancing of TGF-β receptor signaling in non-caveolae microdomains in plasma membrane.

目錄
論文審定書 i
誌謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 vii
縮寫表 ix
第一章　前言 1
一、轉型生長因子β （Transforming Growth Factor-β, TGF-β） 1
二、Lipid raft/Caveolae 4
三、樺木酸（Betulinic acid） 9
第二章　研究目的 11
第三章　材料 12
第四章　實驗方法 16
第五章　結果 23
一、BetA會加強由TGF-β所引起的轉錄反應 23
二、BetA會增強由TGF-β所引起的Smad磷酸化，並且增加p-Smad移至細胞核內的量。 25
三、BetA可促進由TGF-β誘導的Calponin蛋白的表現量 26
四、BetA會增強由TGF-β所誘導之抑制Mv1Lu cell生長 27
五、BetA促使TGF-β受體聚集至non-cavelae區域 28
第六章　討論 43
第七章　總結 46
第八章　參考文獻 47

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