調控分化後的脂肪細胞可以改善肥胖及其相關疾病，包括誘導脂肪細胞形態轉變及減少脂肪細胞數量，然至目前為止，以此改善肥胖方式尚無藥物發展。靈芝屬中國傳統藥物的上品，久用於東方國家。本研究所用松杉靈芝源自桃園羅桂英菌種場的天生靈芝經鑑種而得。本實驗室的體外研究顯示，松杉靈芝乙醇萃取物會影響油滴的大小和粒線體相關的基因表現，同時也發現松杉靈芝乙醇萃取物對3T3-L1前驅脂肪細胞分化後的影響作用類似於羅格列酮，已知羅格列酮是PPAR 激活劑，不但會誘導脂肪的形成也會影響粒線體相關的基因表現和脂肪細胞形態的轉變。因此，本研究的目的是要利用蛋白質體學的方法探討松杉靈芝乙醇萃取物對分化後的3T3-L1有何影響，並找出相關的分子機制。實驗結果顯示，高濃度的松杉靈芝乙醇萃取物會降低3T3-L1分化後的細胞存活率。反之，低濃度的松杉靈芝乙醇萃取物對細胞存活率並無顯著影響。另以Oil-Red O染色實驗發現松杉靈芝乙醇萃取物不會影響分化結果，但會讓油滴變小，這是形態轉變的特徵之一。進一步利用蛋白質體學的技術探討松杉靈芝乙醇萃取物對分化後3T3-L1脂肪細胞是否有“褐變”現象及其可能的機制。結果顯示表現量上升的蛋白質被歸類於細胞代謝者有41%，與蛋白質合成、降解和運輸相關者有17%，與分子伴護蛋白相關者有14%，與其他類蛋白相關的有28%。其中48%的蛋白皆是屬於粒線體蛋白，接著利用西方墨點法進行驗證。綜合上述結果顯示，松杉靈芝乙醇萃取物可能經由誘導氧化代謝作用及mTORC1介導的粒線體和蛋白質生合成作用，進而造成成熟脂肪細胞褐變(browning)的現象。

Targeting the phenotype switch of adipocytes and the removal of adipocytes via reducing cell viability are emerging as potential strategies for the amelioration of obesity and its related disorders. However, there are no potential western drugs reported to be used for these strategies. Discovery of helpful and effective drugs, such as the Traditional Chinese Medicine (TCM), may improve the hazard of obesity. Ganoderma tsugae, a well-known TCM, used in this study is cultivated in Luo-Kui-Yin Fungi Agriculture Farm, Taoyuan, Taiwan. We found that effect of the ethanolic extract of Ganoderma tsugae (GTEE) on 3T3-L1 cell differentiation was similar to that of rosiglitazone (Rosi), a well-known PPAR agonist inducing not only adipogenesis but also mitochondrial biogenesis, resulting in the“browning”of adipocytes in cell culture models. Therefore, we aimed to investigate the potential effects and related mechanisms of GTEE on mature 3T3-L1 adipocytes via proteomics approach. Our previous studies showed that GTEE could reduce the size of lipid droplets without affecting the cell viability of mature 3T3-L1 adipocytes, showing a major feature of browning phenotype switch of adipocytes. To further evaluate the dipocyte “browning” phenomenon and related mechanisms, we performed this study. After treatment with GTEE, up-regulated proteins were identified and categorized into cellular catabolism process (41%), protein biosynthesis, degradation, and transport (17%), molecular chaperone (14%), and others (28%). Especially, 48% (14/29) identified proteins belonged to mitochondrial proteins. Taken together, GTEE may have the potential to cause browning of the mature adipocytes, which may be due to the induction of oxido-reduction metabolism and mTORC1-mediated mitochondria and protein biogenesis.

致謝 I
表目錄 V
圖目錄 VI
縮寫表 VII
中文摘要 IX
Abstract X
第一章 研究緣起與目的 1
第二章 文獻回顧 3
第一節 靈芝 3
第二節　3T3-L1 前驅脂肪細胞 5
第三節　脂肪細胞分化 5
第四節　白色脂肪轉化成類棕色脂肪 6
第三章　研究材料與方法 7
第一節 細胞株來源 7
第二節　松杉靈芝來源 8
第三節　抗體 Antibody 8
第四節　藥品與試劑 8
第五節　主要器材與儀器 12
第六節　實驗方法 13
第四章 實驗結果 23
第一節　松杉靈芝乙醇萃取物之回收率 23
第二節 松杉靈芝乙醇萃取物對3T3-L1成熟脂肪細胞存活率的影響 23
第三節 松杉靈芝乙醇萃取物對於3T3-L1成熟脂肪細胞分化的影響 23
第四節 松杉靈芝乙醇萃取物處理成熟脂肪細胞後蛋白質的表現情形 24
第五節 二維凝膠電泳影像分析與蛋白質分析 24
第六節 粒線體相關蛋白質表現 25
第五章 討論 26
第一節 相關文獻評析與闡釋 26
第二節 實驗結果釋論 27
第六章 結論 33
第七章 參考文獻 34
第八章 圖表 40
第一節 表 40
第二節 圖 44
第九章 附錄 61
附錄一、脂肪堆積型式 61
附錄二、民國103年國人十大死因 62
附錄三、脂肪細胞的分化 63
附錄四、台灣主要八大靈芝分類表 64
附錄五、松杉靈芝(原天生靈芝)之子實體 65
附錄六、脂肪細胞種類 66
附錄七、細胞株處理 67
附錄八、2-Dimensional Gel Electrophresis buffer 72
附錄九、一維等電點聚焦 (IEF) 78
附錄十、西方點墨法 (Western Blotting) 79

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王以均：應用蛋白質體學技術探討松杉靈芝萃取物抑制人類卵巢癌
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