臺灣博碩士論文加值系統

共軛亞麻油酸 (conjugated linoleic acid, CLA) 是一群含18個碳、具共軛雙烯鍵的脂肪酸，為亞麻油酸 (linoleic acid, LA) 的異構物。常見於反芻動物瘤胃中，亦富含於反芻動物的乳、肉及乾酪的乳製品中。近年來研究證明 CLA 具有許多生理功能，包括抑制腫瘤細胞生長、抗動脈硬化、調節免疫反應及調整體脂組成等。本實驗即是使用小鼠之前脂肪細胞為研究模式，評估 CLA 對 3T3-L1 前脂肪細胞及其肥胖分化相關轉錄因子之影響；此外，更透過免疫調節因子的分析模式來探討 CLA 對肥胖之間接抑制作用，故進一步分析肥胖相關激素包括腫瘤壞死因子 (Tumor Necrosis Factor-α, TNF-α) 及 C-反應蛋白 (C-reactive protein, CRP) 之含量。實驗結果顯示，c-9, t-11 CLA 及 t-10, c-12 CLA 在濃度達 50 μg/L 時即會導致 3T3-L1 前脂肪細胞死亡，經由 DNA 片段化、細胞週期分析及Annexin V-FITC/PI 螢光雙染法的分析，皆顯示具有細胞凋亡的特性及現象產生。在脂肪細胞分化方面，隨著 CLA 添加濃度的提高，脂肪分化率及脂肪滴形成的減少也愈加明顯。測量三酸甘油酯 (triglyceride, TG) 的累積量中發現，t-10, c-12 CLA 能明顯降低胞內三酸甘油酯濃度 (p < 0.05)，且具劑量效應，濃度在 25 μg/L 時，約可減少 45% 的三酸甘油酯累積，而同濃度下，c-9, t-11 CLA 則可降低 25% 的三酸甘油酯累積。於脂蛋白脂解酶活性測定上也可發現，不同濃度之兩種 CLA 約在濃度於 25 μg/L 時，抑制效果皆具有顯著差異 (p < 0.05)。在 CLA 抑制 3T3-L1 脂肪細胞分化過程中相關轉錄因子 C/EBPα, PPARγ 及 SREBP-1 基因表現量方面，當 t-10, c-12 CLA濃度在 25 μg/L 時，可明顯降低 C/EBPα, PPARγ 及 SREBP-1基因的表現量 (p < 0.05)，且具劑量效應；另外，c-9, t-11 CLA 於 25 μg/L濃度下，也可降低　C/EBPα 及 PPARγ 基因表現約 20 %，而對　SREBP-1　基因的表現量則無明顯抑制效果。在細胞激素測定方面，將人類單核球細胞株 (THP-1, UMΦ) 及人類肝癌細胞株 (Hep-G2) 以 c-9, t-11 CLA 及 t-10, c-12 CLA 處理，利用細胞酵素連結免疫吸光分析法 (ELISA) 顯示，t-10, c-12 CLA可顯著增加 TNF-α 及 CRP 的分泌量 (p < 0.05)，並具有濃度依附相關性。由以上實驗可看出，相較於兩種型式之 CLA，皆以 t-10, c-12 CLA 較為明顯，故推測於抑制肥胖的效果上，t-10, c-12 CLA 較 c-9, t-11 CLA 為有效。而綜合上述結果說明 CLA 可促進前脂肪細胞凋亡，且可藉由抑制脂肪細胞中與分化相關轉錄因子的基因表現，進一步降低脂肪細胞的分化率及減少胞內脂肪生成；並於免疫相關激素的間接影響上，發現 CLA 可能藉由促進 TNF-α及 CRP 之分泌而達到抗肥胖、抗糖尿病或調節免疫之效果。

Conjugated linoleic acid (CLA) is a collective term for positional and geometric isomers of linoleic acid. CLA occurs naturally in a variety of foods, particularly dairy products and meat from ruminant animals. CLA has been clearly shown to have a number of biological actions: it inhibits carcinogenesis and atherosclerosis; enhanced immune responses, and reduced body fat etc. The objective of this study was to determine the effects of c-9, t-11 and t-10, c-12 isomers of CLA on the differentiation of mouse 3T3-L1 preadipocytes and the production of some cytokines. c-9, t-11 CLA and t-10, c-12 CLA at concentration above 50 μg/L can inhibit the growth of 3T3-L1 preadipocytes. By using DNA fragmentation, cell cycle analysis and Annexin V-FITC/PI assay, showed that CLA can induced preadipocytes apoptosis. On the differentiation of preadipocyte, 3T3-L1 preadipocytes were cultured until confluence, and cells were induced using the differentiation reagents, confluence 3-isobutyl-methylxanthine / Dexamethasone / Insulin (MDI), eight days after the cells were induced for differentiation, by Oil Red O-stained, we found that CLA prevents triacylglycerol accumulation. t-10, c-12 CLA (25 μg/L) showed a 45 % of decreased triglyceride production, and c-9, t-11 CLA (25 μg/L) presented a 25 % of that. Furthermore, we also evaluated the effects of c-9, t-11 and t-10, c-12 isomers of CLA on the expression of C/EBPα, PPARγ and SREBP-1, which are transcriptional factors essential for adipocytes differentiation and lipogenesis, using the RT-PCR technique. Results showed that t-10, c-12 CLA at concentration above 25 μg/L significantly decreased the mRNA expression of C/EBPα, PPARγ and SREBP-1 by 53, 62 and 60 %, respectively. This inhibitory effect was dose-dependent. And c-9, t-11 CLA at concentration above 25 μg/L also decreased the mRNA expression of C/EBPα and PPARγ by 20 %. However, c-9, t-11 CLA presents no inhibition on the expression of SREBP-1. The other evidence is presented indicating that CLA may modulate cellular response to TNF-α and CRP.
These results suggest that CLA can induced preadipocytes apoptosis, inhibit adipogenesis and differentiation through suppressing the associated transcription factors, C/EBPα, PPARγ and SREBP-1. Furthermore, CLA may inhibit 3T3-L1 cell differentiation by interfering with the cytokine regulated pathway.

中文摘要 1
英文摘要 3
壹、前言 5
貳、文獻整理 6
一、共軛亞麻油酸 (conjugated linoleic acid) 6
1. 共軛亞麻油酸之結構與來源 6
2. 共軛亞麻油酸之生理功能 7
2-1. 抗癌特性 7
2-2. 抗動脈硬化 8
2-3. 免疫調節 9
2-4. 體組成之調整 10
二、脂肪細胞 (Adipocytes) 12
1. 肥胖成因與脂肪細胞 12
2. 脂肪細胞之特性與種類 12
3. 脂肪細胞之分化 13
4. 脂肪細胞分化之過程 15
5.脂肪細胞分化之相關轉錄調控基因 17
5-1. SREBP (Sterol regulatory element binding protein) 17
5-1-1. 總論 17
5-1-2. 脂肪組織及脂肪細胞中的 SREBP 17
5-2. CCAAT/促進子結合蛋白質 (CCAAT/enhancer binding proteins) 18
5-2-1. C/EBP 家族與脂肪細胞的分化 18
5-2-2. C/EBPα、C/EBPβ 及 C/EBPδ 19
5-2-3. C/EBP 家族成員間之相互調控 20
5-3. 過氧化體增生活化受體 (Peroxisome proliferator-activated receptor) 20
5-3-1. 總論 20
5-3-2. PPAR 之活化劑 (activators) 與結合子 (ligands) 21
5-3-3. PPARγ與脂肪細胞分化 22
5-3-4. PPARγ 與 C/EBPs 間的相互調控 23
三、肥胖相關之細胞激素 24
1. 腫瘤壞死因子 (Tumor Necrosis Factor α, TNF-α) 24
2. C反應蛋白 (C-reactive protein, CRP) 25
參、實驗設計及材料與方法 26
一、實驗設計大綱 26
二、實驗材料 27
1.材料來源 27
2.細胞株 27
3.藥品 27
4.設備 29
三、實驗項目與實驗方法 30
1. 細胞培養 30
2. 脂肪細胞之分化培養 31
3. 油紅 O 染色 (Oil red O stain) 31
4. 細胞內三酸甘油酯 (Triglyceride) 含量之分析 31
5. 脂蛋白脂解酶 (Lipoprotein lipase protein) 含量之分析 32
6. 細胞存活率 (MTT assay) 32
7. DNA 片段化之測定 (DNA Fragmentation Test) 32
8. Annexin V-FITC/PI 雙染法 33
9. 細胞週期分析 33
10. 反轉錄-聚合酶連鎖反應 34
10-1. 抽取 3T3-L1 細胞 total RNA 34
10-2. 反轉錄反應 (RT) 34
10-3. 聚合酶連鎖反應 (PCR) 35
10-4. DNA 電泳與膠片定量分析 35
11. TNF-α 濃度測定 35
12. CRP 濃度測定 36
13. 統計分析 36
肆、結果與討論 37
一、共軛亞麻油酸對 3T3-L1 前脂肪細胞增生之影響 37
二、共軛亞麻油酸對 3T3-L1 前脂肪細胞致死現象之探討 37
1. 螢光雙染法 37
2. DNA 片段化測定 38
3. 細胞週期分析 38
三、共軛亞麻油酸對 3T3-L1 脂肪細胞型態之影響 39
1. 3T3-L1 脂肪細胞培養與分化 39
2. CLA對 3T3-L1 脂肪細胞型態之影響 39
四、共軛亞麻油酸對脂肪細胞胞內三酸甘油酯含量之影響 40
五、共軛亞麻油酸對脂肪細胞胞內脂蛋白脂解酶活性之影響 40
六、共軛亞麻油酸對 3T3-L1 分化相關轉錄因子之影響 41
1.分析方法之建立 41
2. CLA 對脂肪細胞分化轉錄因子表現的影響 42
七、共軛亞麻油酸對腫瘤壞死因子分泌之影響 43
八、共軛亞麻油酸對C-反應蛋白分泌之影響 44
伍、結論 46
陸、參考文獻 48
柒、圖表 64
捌、附錄 87

圖表目錄
表1、PCR 引子設計及序列產物長度……………………………………….64
圖 1. c-9, t-11 CLA及t-10, c-12 CLA 對3T3-L1前脂肪細胞增生之影響...65
圖 2. c-9, t-11 CLA及t-10, c-12 CLA 作用於3T3-L1前脂肪細胞之細胞形態改變情形………………………………………………………………...66
圖 3. c-9, t-11 CLA及t-10, c-12 CLA 作用於3T3-L1前脂肪細胞之 DNA 片段化情形………………………………………………………………...67
圖 4. c-9, t-11 CLA及t-10, c-12 CLA 作用於3T3-L1前脂肪細胞之 sub-G1 期細胞百分比……………………………………………………...……68
圖 5. c-9, t-11 及t-10, c-12 CLA 對 3T3-L1細胞脂質形成累積之影響.…69
圖6. c-9, t-11 及t-10, c-12 CLA 對 3T3-L1細胞中三酸甘油酯累積之影響………………………………………………………………………...70
圖 7. c-9, t-11 及t-10, c-12 CLA 對 3T3-L1細胞中脂蛋白脂解酶活性之影響……………………………………………………………………..….71
圖 8. PPARγ、SREBP-1、C/EBPα 與 β-actin 之專一性引子經反轉錄聚合酶連鎖反應所增殖之 cDNA 片段……………………………………….72
圖 9. c-9, t-11 CLA 對分化第八天之 3T3-L1細胞中 SREBP-1 mRNA表現量之影響……………………………………………...…………………73
圖 10. c-9, t-11 CLA 對分化第八天之 3T3-L1細胞中 C/EBPα mRNA表現量之影響…………………………………………………………..…….74
圖 11. c-9, t-11 CLA 對分化第八天之 3T3-L1細胞中 PPARγ mRNA表現量之影響……………………………………………………..…………….75
圖 12. t-10, c-12 CLA 對分化第八天之 3T3-L1細胞中 SREBP-1 mRNA表現量之影響…………………………………...…………...…………...76
圖 13. t-10, c-12 CLA 對分化第八天之 3T3-L1細胞中 C/EBPα mRNA表現量之影響……………………………………………………………….77
圖 14. t-10, c-12 CLA 對分化第八天之 3T3-L1細胞中 PPARγ mRNA表現量之影響…………………………….…………………………………78
表 2. c-9, t-11 與 t-10, c-12 CLA 對3T3-L1 脂肪細胞分化相關轉錄因子表現之抑制率……………………………………………………………...79
圖 15. c-9, t-11 CLA及t-10, c-12 CLA 對人類單核球細胞株增生之影響…….…………………………………………………………………80
圖 16. CLA作用於人類單核球細胞株之腫瘤壞死因子釋出量.................... 81
圖 17. c-9, t-11 CLA及t-10, c-12 CLA 對人類巨噬細胞增生之影響….…..82
圖 18. CLA作用於人類巨噬細胞之腫瘤壞死因子釋出量.............................83
圖 19. c-9, t-11 CLA及t-10, c-12 CLA 對人類肝癌細胞株增生之影響.......84
圖 20. CLA作用於人類肝癌細胞株之C-反應蛋白釋出量............................85
圖 21. CLA作用之可能機制..................................................................……...86
附錄
附錄 1、不飽和十八碳脂肪酸進行生物氫化作用之主要路徑…………..…87
附錄 2、共軛亞麻油酸生物合成之路徑……………………………………..88
附錄 3、實驗室培養前脂肪細胞分化的過程…...……………………………89
附錄 4、3T3-L1 前脂肪細胞內基因於細胞分化過程中的表現……………90
附錄 5、PPARγ 與 C/EBP 家族成員間的相互調控 ………………………91

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