臺灣博碩士論文加值系統

肥胖人口的遽增，已是許多國家所面臨相當嚴重的健康議題，不但造成生活品質下降，醫療花費的增加，更是許多疾病的危險因子。而肥胖發生的主要機制在於脂肪的形成與堆積，因此抑制脂肪細胞的新生為預防肥胖的首要關鍵。至今已有許多研究證實天然植物或中草藥萃取物中含有抑制脂肪細胞新生及脂肪累積的活性成份，被視為治療與預防肥胖的一個新療法，在本實驗中，利用3T3-L1細胞模式，篩選出石菖蒲精油與花椒精油具有抑制脂肪細胞分化的作用，並且鑑定出其中有效成份分別為-asarone及methyl cinnamate。因此，本研究主要目的為探討-asarone及methyl cinnamate對脂肪細胞新生的影響以及可能的分子作用機制。結果顯示-asarone及methyl cinnamate能有效降低脂肪細胞內三酸甘油酯的累積，-asarone可降低脂肪細胞分化相關轉錄因子C/EBP、PPARγ及C/EBP的表現，並且在細胞分化早期顯著抑制phospho-ERK的蛋白質表現。而methyl cinnamate則抑制了C/EBP、SREBP-1、PPARγ及C/EBP的表現，並且造成PPARγ下游基因aP2、adiponectin表現以及GPDH酵素活性下降，此外methyl cinnamate還具有拮抗PPARγ的轉錄活性，而methyl cinnamate則是透過活化CaMKK2-AMPK訊號傳遞途徑抑制脂肪細胞基因的表現及脂質的合成，達到抑制脂肪細胞分化的作用。綜合以上結果，本實驗證實-asarone及methyl cinnamate具有抑制脂肪細胞形成的活性，並進一步了解其作用機制，為未來治療肥胖或是肥胖相關代謝性疾病的藥物開發奠定基礎。

The incidence of obesity dramatically rising worldwide and is a major contributor to the global burden of metabolic disorders. Excessive accumulation of adipose tissue mass contributes to the development of obesity. Adipogenesis, combines with adipocyte differentiation and proliferation, is an important mechanism that leading to the increase of adipocytes. Therefore, inhibition of adipogenesis is the key way to prevent obesity. Several studies have been discussed the therapeutic potentials of essential oil and their volatile components, including anti-obesity. In this study, we used 3T3-L1 preadipocytes to find the anti-adipogenic effect of essential oils and the active components. Acorus calamus and Zanthoxylum armatum essential oils showed the ability to suppress adipogenesis in 3T3-L1 preadipocytes. Two active compounds, -asarone and methyl cinnamate, were identified, respectively. -Asarone significantly reduced triglyceride accumulation during adipocyte differentiation in a dose-dependent manner. The expressions of adipocyte transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein  (C/EBP) and C/EBP were down-regulated after treatment with -asarone. Moreover, ERK activation known to regulate the early phase of adipogenesis was inhibited by treatment with -asarone. Methyl cinnamate markedly suppressed triglyceride accumulation associated with down-regulation of adipogenic transcription factors expression, including sterol regulatory element binding protein-1 (SREBP-1), C/EBP, PPARγ and C/EBP. Additionally, methyl cinnamate-inhibited PPARγ activity and adipocyte differentiation were partially reversed by PPARγ agonist, troglitazone. Furthermore, methyl cinnamate stimulated Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), phospho-AMP-activated protein kinase (AMPK) and phospho-ERK expression during adipogenesis. The results suggested methyl cinnamate has anti-adipogenic activity through mechanisms mediated, in part, by CaMKK2-AMPK signaling pathway in 3T3-L1 cells. Overall, our studies suggested -asarone and methyl cinnamate exhibited anti-adipogenic activity and have the potential to be developed as an anti-obesity agent and a treatment for obesity-related metabolic syndrome.

中文摘要 4
Abstract 5
第一章、緒論 7
一、肥胖症介紹 7
二、脂肪組織(adipose tissue) 8
三、脂肪細胞的新生(adipogenesis) 9
1. 調控脂肪細胞新生的轉錄因子 9
2. Insulin與MAPK訊號傳遞途徑 11
3. AMPK訊號傳遞途徑 12
四、3T3-L1脂肪細胞模式 13
五、精油與植物化合物 14
六、治療肥胖的藥物 15
七、石菖蒲精油(Acorus calamus)與-asaone 16
八、花椒精油(Zanthoxylum armatum)與methyl cinnamate 17
九、研究動機與目的 18
十、實驗架構與策略 18
第二章、材料與方法 20
一、實驗材料 20
1. 細胞培養 20
2. 化學藥品 20
3. 抗體 21
4. 儀器設備 21
二、實驗方法 22
1. 3T3-L1細胞培養與分化 22
2. Oil red O染色 22
3. 三酸甘油酯含量分析 22
4. 細胞存活率分析(MTT assay) 23
5. RNA萃取與即時定量PCR (quantitative real time-PCR) 24
6. 蛋白質萃取與定量 24
7. 西方墨點法(Western blot) 25
8. 氣相層析質譜儀分析(GC/MS analysis) 26
9. 矽膠管柱層析分離 26
10. 高效能液相層析儀分析(high performance liquid chromatography； HPLC) 27
11. GPDH活性測定 27
12. PPAR活性測定 28
13. 統計分析 28
第三章、結果 29
一、抑制脂肪細胞分化之植物精油篩選結果 29
二、石菖蒲精油對於脂肪細胞分化的影響 29
三、石菖蒲精油成分分析及有效成分分離 30
四、-Asarone對於脂肪細胞分化的影響 31
五、-asarone對於C/EBP、C/EBP及PPAR表現的影響 31
六、-asarone對於ERK蛋白表現的影響 31
七、花椒精油對脂肪細胞分化的影響 32
八、花椒精油主要活性成分分析 32
九、Methyl cinnamate對脂肪細胞分化之影響 33
十、Methyl cinnamate對GPDH活性的影響 33
十一、Methyl cinnamate對脂肪細胞專一指標因子表現的影響 34
十二、Methyl cinnamate對於PPAR轉錄活性的影響 35
十三、Methyl cinnamate對於CaMKK2-AMPK訊號傳遞途徑的影響 35
十四、Methyl cinnamate對於ERK及C/EBP表現的影響 36
十五、Methyl cinnamate結構類似化合物對於脂肪新生的影響 37
第四章、討論 38
一、植物性化合物治療肥胖的潛力 38
二、-Asarone抑制脂肪細胞分化作用機制 39
三、Methyl cinnamate抑制脂肪細胞分化作用機制 40
四、PPAR活性調控的重要 42
五、物質結構和抑制脂肪新生功能相關性 43
六、-Asarone與Methyl cinnamate安全性探討 44
七、抑制脂肪細胞分化治療減肥研究的爭議 45
八、總結 46
第五章、表格與附圖 48
表一、Real time-PCR引子序列 48
表二、RT-PCR引子序列 49
表三、石菖蒲精油主要成分列表 50
表四、石菖蒲精油Fraction 3主要成分列表 51
表五、花椒精油主要成分列表 52
表六、精油成分對於3T3-L1細胞內三酸甘油酯累積的影響 53
圖一、石菖蒲精油抑制3T3-L1細胞內三酸甘油酯的累積 54
圖二、石菖蒲精油GC/MS分析圖譜 55
圖三、HPLC純化asarones分析圖譜 56
圖四、化合物結構圖 57
圖五、-Asarone對3T3-L1細胞分化的影響之oil red o染色圖 58
圖六、-Asarone對3T3-L1細胞內三酸甘油酯含量的影響 59
圖七、在3T3-L1細胞分化過程中-Asarone對於C/EBP表現的影響 60
圖八、在3T3-L1細胞分化過程中-Asarone對於C/EBP及PPAR表現的影響 61
圖九、在3T3-L1細胞分化過程中-Asarone對於ERK蛋白表現的影響 62
圖十、花椒精油對3T3-L1細胞分化的影響 63
圖十一、花椒精油GC/MS分析圖譜 64
圖十二、Methyl cinnamate對於3T3-L1脂肪細胞存活率的影響 65
圖十三、Methyl cinnamate對於3T3-L1脂肪細胞分化的影響 66
圖十四、Methyl cinnamate對於3T3-L1細胞內油滴累積及GPDH活性的 影響 67
圖十五、Methyl cinnamate抑制脂肪細胞PPAR、C/EBP及SREBP-1基因表現 68
圖十六、Methyl cinnamate對於3T3-L1細胞aP2及adiponectin基因表現的影響 69
圖十七、Methyl cinnamate對於3T3-L1細胞PPAR、C/EBP及resistin蛋白質表現的影響 70
圖十八、Methyl cinnamate對於3T3-L1細胞PPAR轉錄活性的影響 71
圖十九、Methyl cinnamate對於CaMKK2-AMPK訊號途徑蛋白表現的影響 72
圖二十、CaMKK2抑制劑STO-609緩解methyl cinnamte抑制脂肪細胞分化的作用 73
圖二十一、Methyl cinnamate對於phospho-ERK及C/EBP蛋白表現的影響 74
圖二十二、Methyl cinnamate結構類似物質對脂肪細胞油滴累積的影響 75
圖二十三、Methyl cinnamate抑制脂肪細胞分化可能的調控機制 76
附圖一、脂肪細胞新生過程示意圖 77
附圖二、脂肪細胞分化過程及轉錄因子的調控 78
附圖三、Insulin調控脂肪細胞新生傳遞途徑 79
附圖四、MAPK調控脂肪細胞新生傳遞途徑 80
附圖五、AMPK調控脂肪細胞分化及脂質合成傳遞途徑 81
附圖六、活化CaMKK2-AMPK訊號傳遞抑制脂肪細胞分化可能調控機制圖 82
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