- 學位論文
Disintegrin對脂肪細胞新生作用之探討
Regulatory effect of disintegrin on adipogenesis
摘要
脂肪新生(adipogenesis)在肥胖的發展上扮演了很重要的角色，其過程包括前驅脂肪細胞的移動、附著、增生、生存及分化為成熟的脂肪細胞，而這些機能都和細胞上integrin的調控有關，於是我們利用由蛇毒中所純化出的disintegrin－trigramin、triflavin、rhodostomin－來探討它們對脂肪新生的影響。由實驗結果發現，disintegrin會造成初級前驅脂肪細胞及3T3-L1細胞株的脫落，並對focal adhesion產生影響，包括p-FAK、FAK表現減少及改變vinculin的聚集和actin骨架的排列。另外前驅脂肪細胞的存活率會隨著加入的disintegrin濃度的增加而有明顯的減少，利用流式細胞儀可發現細胞有凋亡的情形，而DAPI染色更可看到細胞核有濃縮的現象；分析和細胞凋亡有關的蛋白變化，也看到以disintegrin處理過的細胞其Bax的表現及caspase 3的活化態皆呈現增加的趨勢。若將培養皿舖上各種胞外基質時，disintegrin會抑制細胞附著於osteopontin及vitronectin，並使已貼附在osteopontin及vitronectin上的細胞產生脫落而降低存活率，但對於fibronectin、laminin、collagen的附著則不受影響。另外我們發現，前驅脂肪細胞的移行能力也會在disintegrin的作用之下受到抑制。當以IBMX、dexamethasone、insulin誘發前驅脂肪細胞分化時，同時加入disintegrin處理後，同樣也會造成細胞的脫落及減少存活率，最後使分化中的細胞走向凋亡，於是在經過9~10天的分化後，在分化初期經過disintegrin處理的細胞其分化程度明顯地受到抑制，利用triglyceride測定及以RT-PCR分析成熟脂肪細胞特定表現的基因(PPARγ和leptin)也都再次證明分化成熟的細胞數量在disintegrin作用下是減少的。綜合以上結果，disintegrin可以抑制脂肪新生的作用而可能有機會應用在肥胖的治療上。
並列摘要
Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Because these biological functions are all related to the regulation of integrin, here we used these snake venom-derived disintegrins including trigramin, triflavin and rhodostomin to study the role of integrin in adipogenesis. It was found that disintegrin caused the detachment of primary cultured preadipocyte and 3T3-L1 cell line. Furthermore, disintegrins also inhibited cell focal adhesion including decrease of the expression of FAK and FAK phosphorylation, reassembly of vinculin, and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after disintegrin treatment in a dose-dependent manner. The results of flow cytometric analysis showed that disintegrins induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase 3 were detected following disintegrin treatment. Snake venom disintegrin inhibited the adhesion of preadipocytes to immobilized vitronectin and osteopontin but not to fibronectin, laminin and collagen. Furthermore, migration of preadipocytes was also inhibited by disintegrin treatment. Addition of IBMX, dexamethasone and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with disintegrins during the initial 3 days also detached the differentiating cells and induced apoptosis. The disintegrin-treated cells showed less mature adipocytes following 9~10 days of differentiating period. The triglyceride content and gene expression of PPARγ and leptin also decreased in response to the treatment of disintegrin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.