臺灣博碩士論文加值系統

靈芝為著名藥用真菌，具有許多藥理作用，在亞洲國家具長久使用歷史，以促進健康為目的。目前已知脂肪細胞的分化(脂肪生成)在肥胖的發展過程中和調控肥胖的平衡扮演著重要的角色。本實驗所使用的靈芝於臺灣桃園栽培，為台灣常見靈芝種類之一。先前的研究發現，低劑量GTEE會促進脂肪細胞的分化及小油滴的形成，但並不會對脂肪細胞造成危害，小且多的油滴均為脂肪細胞褐變的重要特徵。故本研究進一步探討GTEE影響脂肪細胞分化的相關機制。利用蛋白質體學技術，探討於脂肪細胞分化過程中以GTEE處理後其蛋白質圖譜的差異，進行識別及建立相關分子機制。選擇其中上表達的蛋白質進行鑑定，結果主要可分為分解代謝(67%)，輔助蛋白摺疊蛋白(10%)及其他(23%)等三大類蛋白。有趣的是，於上調控的蛋白質中參與氧化還原狀態調節及粒線體內蛋白質分別佔有33%及47%。根據蛋白質鑑定結果及進一步利用西方墨點法與測定NAD+/NADH含量進行驗證，結果顯示GTEE在脂肪細胞分化過程中具誘導脂肪細胞褐變之功效。綜合上述研究結果可知GTEE具有促進3T3-L1脂肪細胞分化之生理功效並可調控能量代謝。

Ganoderma or Lingzhi has been used for health promotion in Asian countries for many centuries. Adipocyte differentiation (adipogenesis) is a key aspect to obesity development. Our pilot studies have found that ethanolic extract of Ganoderma tsugae (GTEE), one of the most common species of Lingzhi cultivated in Taoyuan, Taiwan, has potential in affecting adipogenesis in murine 3T3-L1 cells. However, the relevant molecular mechanism of GTEE on adipogenesisis yet to be explored. Here, a proteomics approach was employed to elucidate the GTEE-mediated protein expression profiles during adipogenesis and to unravel the relevant molecular mechanisms. Our previous studies showed that low dose of GTEE could influence 3T3-L1 adipocyte differentiation and small lipid droplet (LD) formation without significantly affecting cell viability. Small and multiple LD formation were important features of browning-like adipocytes. By using the proteomics strategy our results revealed that the GTEE-mediated up-regulated proteins were identified and categorized into cellular catabolism process (67%), molecular chaperone (10%), and others (23%). Interestingly, the up-regulated proteins involved in redox state regulation and mitochondrial proteins were 33% and 47%, respectively. Based on the protein profiles followed by further western blotting and NAD+/NADH quantitation assay validation, GTEE-mediated cellular catabolism process leading to the browning-like adipocyte differentiation was proposed. Taken together, these findings may suggest that GTEE can enhance the adipogenesis and modulate the energy metabolism in 3T3-L1 cells.

致謝 I
目錄 II
圖目錄 IV
縮寫表 V
中文摘要 VII
Abstract VIII
第一章　研究緣起與目的 1
第二章　文獻回顧 3
第一節、靈芝（Ganoderma; Reishi; Lingzhi） 3
第二節、脂肪細胞 4
第三節、脂肪細胞培養與分化 5
第四節、蛋白質體學 7
第三章 研究材料與方法 9
第一節、實驗材料與操作儀器 9
第二節、實驗方法 14
第四章 實驗結果 28
第一節、松杉靈芝乙醇萃取物（GTEE）對分化中3T3-L1脂肪細胞之影響 28
第二節、GTEE對3T3-L1脂肪細胞分化的影響 28
第三節、 GTEE對分化中的3T3-L1脂肪細胞蛋白質表現的二維電泳、膠片分析、質譜分析及資料庫比對結果 29
第四節、 GTEE對分化中的3T3-L1脂肪細胞內粒線體及能量相關蛋白之影響 31
第五節、 GTEE對分化中的3T3-L1脂肪細胞內NADH含量及NAD+/NADH Ratio之影響 32
第六節、 GTEE影響分化中3T3-L1脂肪細胞之可能代謝路徑 32
第五章 討論 34
第六章 結論 39
第七章 參考文獻 40
第八章 圖表 45
第九章 附錄 61
附錄一、動物體內3種脂肪細胞間的比較 61
附錄二、白色脂肪細胞分化圖 62
附錄三、脂肪細胞分化各階段一覽圖 63
附錄四、松杉靈芝（取自天生靈芝）子實體 64
附錄五、松杉靈芝乙醇萃取物（GTEE）萃取方法 65
附錄六、試劑及緩衝液配置 66
附錄七、一維等電點聚焦設定條件 73

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