臺灣博碩士論文加值系統

Trimucrin是本實驗室從台灣龜殼花毒蛇線基因庫所選殖，並以經酵母菌成功的表達出具抗血小板凝集活性的基因表達蛋白。而這表達蛋白經本實驗室證實除了在活體外具有抗血小板凝集活性，在活體內也具有減重的效果。在減重實驗中，腹部注射Trimucrin能夠有效的使肥胖的大鼠體重下降。就生理而言，體重過重的動物，牠們脂肪組織的脂肪細胞不僅有增殖還有肥大的情形。而這種改變與細胞上不同的黏著蛋白具有著相當的關連性。在本實驗中是以3T3-L1細胞來作為研究分子機制的細胞模式。我們是以固醇酯培養液誘發脂質堆積的3T3-L1細胞來研究Trimucrin可能引起的脂質分解作用的機制，以及可能參與其中的訊息傳遞路徑。研究結果也證實了，Trimucrin會藉由活化HSL來分解三酸甘油酯，使得細胞培養液中的甘油釋出量增加，導致脂質堆積的3T3-L1細胞中的脂質減少使脂肪小滴減少或消失，證實了Trimucrin會誘發3T3-L1細胞脂質分解的功效。而誘發脂質分解的功效可能是透過Trimucrin與細胞表面的整合素結合所造成。因為這現象在加入了整合素的拮抗劑GRGDTP後會被完全的抑制。此外，在加入PKA與ERK的抑制劑之後也發現HSL的活化完全被阻斷，顯示Trimucrin與整合素結合的所刺激的反應是通過PKA與ERK的訊息傳遞路徑所造成。

Trimucrin, a disintegrin protein with potential anti-platelet aggregation activity, was isolated from snake venom of Trimeresurus mucrosqumatus (Taiwan habu) and successfully expressed in Pichia pastoris. The expressed protein demonstrated not only with anti-platelet aggregation activity in vitro but also with body weight-reduction effect in vivo. In the weight-reduction assay, trimucrin reduced the body weight of rats effectively in reverse to their body weight. Physiologically, in overweighed animals, their adipose tissues show not only hyperplasia but also hypertrophy in cellular characteristic and the changes of adipose tissue were highly associated with integrin receptor differentiation. In this study, 3T3-L1 adipocyte cells could provide a useful cellular model in the investigation of molecular mechanisms. In this proposal, we examined the molecular mechanism of trimucrin on lipolysis in sterol ester (SE)-induced lipid accumulated 3T3-L1 cells and to determine the possible signaling mechanism involved. Our results demonstrated that the trimucrin treated 3T3-L1 cells resulted in the reduction in lipid droplets which were characterized by the reduction of lipid accumulation, the increase of HSL activity and the release of glycerol into the culture cell supernatant. Our study also strongly suggested that the trimucrin treated cells induced a lipolysis effect in 3T3-L1 cells. However, this Trimucrin-induced lipolysis effect might be induced through the integrin receptors binding. And these phenomena were highly supported by the complete inhibition of an integrin antagonist GRGDTP in this study. In addition, the integrin binding signaling pathway were confirmed by the downstream PKA and ERK pathway by specific inhibitors of protein kinase A (PKA) and extracellular signal-regulated kinase (ERK).

目錄…………………………………………….………………………….. I
圖次目錄…………………………………………………….……….....…… II
中文摘要……………………….……...……………………………...…...... IV
英文摘要……………………….…………………………………………. V
第一章 緒論…………………….……………………………………….... 1
第二章 材料與方法………….………………………………………...…. 13
第三章 結果…………….…………………………………………...……. 38
第四章 討論…………….……………………………………………...…. 44
第五章 結論………….……………………………………………...……. 47
第六章 參考文獻……………..………………………………………...… 63
附錄

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