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研究生:郭靜如
研究生(外文):Jing-Ru Kuo
論文名稱:中國橄欖甲醇萃取-乙酸乙酯區分層對3T3-L1葡萄糖攝取之功效與機制探討
論文名稱(外文):The mechanism study underlying glucose uptake mediated by Canarium album in 3T3-L1 Adipocytes
指導教授:謝淑貞謝淑貞引用關係
指導教授(外文):Shu-Chen Hsieh
口試委員:黃智興郭靜娟羅翊禎姜安娜
口試委員(外文):Tze-Sing HuangChing-Chuan KuoYi-Chen LoAn-Na Chiang
口試日期:2015-07-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:97
中文關鍵詞:中國橄欖葡萄糖攝取3T3-L1脂肪細胞
外文關鍵詞:Chinese oliveglucose uptake3T3-L1 adipocyte
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根據衛福部最新數據統計台灣糖尿病在十大死因中高居第五名,且罹病人口有逐漸攀升的趨勢,其主要的病因為肥胖所造成的胰島素阻抗。胰島素阻抗是由於周邊組織對於胰島素的反應不敏感,導致葡萄糖無法被周邊組織有效攝取利用。而其中脂肪組織扮演重要角色,過多的脂肪堆積容易造成胰島素阻抗。中國橄欖含有豐富的多酚類、黃酮類之衍生物,具有抗發炎、抗菌、抗氧化及抗癌細胞增生等功效。本實驗室在動物實驗中發現中國橄欖甲醇萃取-乙酸乙酯區分層 (COE) 具有緩減糖尿病的功能,因此本研究將以3T3-L1小鼠脂肪細胞模式探討其機轉。葡萄糖攝取試驗結果顯示COE可以刺激成熟脂肪細胞對2-NBDG的攝取,並在胰島素阻抗的情況下亦可增加2-NBDG的攝取量,而COE的給予可增加3T3-L1之Glucose transpoter 1 (GLUT1) 基因表現量且具有劑量效應,此外利用PI3K、GLUT及AMPK抑制劑均無法完全阻斷COE所促進的葡萄糖攝取。而COE對於3T3-L1 AMPK活性調節並不明確,僅可確定COE可促進AKT活性表現,且可稍微增加GLUT4轉位細胞膜上的量。
由此可推論COE對於刺激脂肪細胞葡萄糖攝取可能不同於PI3K及AMPK路徑。而COE是透過何種訊息傳導路徑來增加GLUT1基因表現及其對於葡萄糖攝取的影響則仍需更一步的研究與探討


Diabetes ranks the fourth leading cause of death in Taiwan, according to the latest report by Ministry of Health and Welfare, and the prevalence is going up. The major cause of diabetes is obesity-induced insulin resistance, which is reduced glucose uptake by peripheral tissues due to compromised sensitivity to insulin as a result of excess lipid accumulation in adipose tissues. Chinese olive is rich in bioactive compounds such as phenolics and flavonoids, and shows anti-inflammatory, antibacterial, anti-oxidative, and anti-proliferative effects. Previous in vivo studies indicate that the ethyl acetate fraction of methanol extract of Chinese olive (COE) ameliorates diabetes. The present study then aims to investigate the underlying mechanism for COE’s anti-diabetic effects using 3T3-L1 mouse adipocytes. Results of glucose uptake test show COE stimulates the uptake of 2-NBDG by mature adipocytes, and such effect is not ablated in the presence of insulin resistance, nor completely abolished by the use of inhibitors for PI3K, GLUT, or AMPK. In addition, treatment of COE can dose-dependently increase transcription of the glucose transporter 1 gene in 3T3-L1. Its regulatory role in AMPK activity in 3T3-L1 is not certain, but it is clear that COE can stimulate the expression of AKT and increase GLUT4 translocation to plasma membrane.
Therefore it is postulated that the mechanism behind COE’s stimulatory effect on glucose uptake of adipocytes is different from PI3K and AMPK signaling pathways. Further study is needed to understand the underlying mechanism of how COE increases GLUT1 gene expression and glucose uptake.



目錄
口委審定書ii
謝誌iii
縮寫表xi
摘要xii
Abstractxiii
前言1
第一章 文獻回顧2
第一節 血糖恆定2
1.1.1 血糖恆定2
1.1.2 胰島素2
1.1.3 葡萄糖攝取路徑4
1.1.4 葡萄糖轉運蛋白6
第二節 糖尿病致病機轉9
1.2.1 糖尿病定義9
1.2.2 胰島素阻抗10
1.2.3 慢性發炎13
1.2.4 脂質代謝異常13
1.2.5 糖尿病治療藥物14
第三節 3T3-L1脂肪細胞模式15
1.3.1 脂肪細胞與血糖15
1.3.2 3T3-L1脂肪細胞 簡介18
1.3.3 AMPK與脂肪細胞19
第四節 中國橄欖22
第二章 研究目的與實驗架構26
第一節 研究目的26
第二節 實驗架構27
第三章 實驗材料與方法29
第一節 實驗材料29
3.1.1 實驗試劑與設備29
3.1.2溶液配製33
第二節 實驗方法35
2.2.1 中國橄欖甲醇萃取-乙酸乙酯區分層之樣品製備 (Preparation of olive extract sample)35
2.2.2 3T3-L1細胞培養與分化 (3T3-L1 cell line culture and differentiation)36
2.2.3 油紅染色 (Oil red O staining)37
2.2.4 2-NBDG 葡萄糖攝取試驗 (2-NBDG glucose uptake assay) 37
2.2.5 總細胞蛋白萃取法 (Total cell protein extraction)37
2.2.6 細胞膜分層分離 (Plasma membrane fraction separation)38
2.2.7 蛋白質定量 (Protein quantification)38
2.2.8 西方墨點法 (Western blotting)38
2.2.8即時半定量mRNA 表現 (Real-time semi-quantification PCR)39
2.2.9統計方法 (Statistics)41
第四章 結果與討論42
第一節 脂肪代謝42
4.1.1 3T3-L1分化模式建立42
4.1.2 中國橄欖甲醇萃取-乙酸乙酯區分層對3T3-L1前脂肪細分化影響49
第二節 葡萄糖攝取60
4.2.1 以3T3-L1建立2-NBDG葡萄糖攝取模式60
4.2.2中國橄欖甲醇萃取-乙酸乙酯區分層刺激葡萄糖攝取63
4.2.3 在胰島素誘導胰島素阻抗模式下之葡萄糖攝取刺激作用65
第三節 中國橄欖甲醇萃取-乙酸乙酯區分層刺激葡萄糖攝取的機制探討68
4.3.1 中國橄欖甲醇萃取-乙酸乙酯區分層活化AMPK 及AKT的效果68
4.3.2不同訊息傳導路徑抑制劑對中國橄欖甲醇萃取-乙酸乙酯區分層提升葡萄糖攝取之影響 74
第四節 中國橄欖甲醇萃取-乙酸乙酯區分層對於GLUT的調控76
4.4.1 GLUT mRNA表現調控76
4.4.2 COE對於3T3-L1脂肪細胞PPAR Ƴ基因表現調控77
4.4.3 GLUT蛋白質表現79
第六章 結論83
第七章 參考文獻參考84


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