臺灣博碩士論文加值系統

肥胖是已開發與開發中國家之主要公共衛生議題，已知其是許多疾病之危害因子，例如：糖尿病、癌症、心臟病與高血壓。過去研究指出，深層海水可抑制3T3-L1脂肪細胞之存活並減少動物體重增加，但深層海水對於脂質合成之抑制相關代謝機制尚不明確。因此本研究將進一步利用細胞及動物模式，探討深層海水在脂質生成之作用與其分子機轉。內容分成兩部分：(一) 以體外細胞試驗模式釐清深層海水之抗肥胖作用與分子機轉；(二) 以體內動物試驗模式探討深層海水抑制高脂飲食誘導肥胖大鼠之影響及其分子機轉。(一) 在細胞試驗部分，結果顯示深層海水對前脂肪細胞之存活率與脂肪細胞之油紅染色並不具有影響 (p>0.05)，但是其可降低脂肪細胞胞內三酸甘油酯含量與甘油-3磷酸去氫酶活性。在基因表現上，深層海水具有影響脂肪細胞分化 (PPARγ、SREBP-1c、C/EBPα與C/EBPβ)、脂質生合成 (ACC、SREBP-1a與HMG-CoA reductase) 與脂肪細胞激素 (Leptin、IL-6、MCP-1、PAI-1與CRP) 之基因表現，以及提升脂質分解與氧化 (ATGL、HSL、CPT-1與ACO) 之基因表現。由此可知，深層海水可透過影響脂質生合成與脂肪分解作用，而抑制3T3-L1脂肪細胞胞內三酸甘油酯與甘油-3磷酸去氫酶活性。(二) 在動物試驗部分，結果顯示，高脂飲食給予深層海水之組別在體重 [DSW組 (1X-5X)]、腎周圍脂肪組織 [DSW組 (1X-5X)]、肝臟 [DSW組 (2X-5X)] 與副睪脂肪組織 [DSW組 (3X-5X)] 之增加較高脂飲食組為低 (p<0.05)。高脂飲食給予深層海水之組別，在肝臟三酸甘油酯 [DSW組 (2X-5X)] 與膽固醇 [DSW組 (3X-5X)] 含量之增加較高脂飲食組為低 (p<0.05)。在血清三酸甘油酯、胰島素與肝指數ALT含量上，高脂飲食給予DSW (1X-5X) 組別可顯著較高脂飲食組為低 (p<0.05)。高脂飲食給予深層海水組別可顯著降低血糖 [DSW組 (2X-5X)]、總膽固醇 [DSW組 (1X、2X與5X)]、游離脂肪酸 [DSW組 (1X、3X與5X)] 與AST [DSW組 (5X)] 之含量 (p<0.05)。在肝臟和脂肪組織切片中，高脂飲食給予深層海水組別可顯著降低其油滴累積 [DSW組 (3X-5X)] 與脂肪組織內較大油滴 [DSW組 (1X-5X)] 的產生。此外，高脂飲食給予深層海水可提升糞便總脂質 [DSW組 (1X-5X)]、三酸甘油酯 [DSW組 (2X-5X)] 與膽固醇 [DSW組 (2X-5X)] 之排出。而高脂飲食給予深層海水之組別可顯著降低肝臟malondialdehyde (MDA) [DSW (1X與3X)] 以及提升glutathione reductase (GRd) [DSW組 (3X-5X)] 之含量。在基因表現上，深層海水組會提升肝臟中PPARα、AMPK、ACO與CPT-1之基因表現。此外，會提升脂肪組織脂質氧化分解 (ATGL、HSL、ACO與CPT-1)，以及降低脂肪細胞激素 (TNF-α、PAI-1與resistin) 之基因表現。綜合以上結果證實，深層海水可降低高脂飲食所誘導肥胖大鼠之血脂異常、脂肪肝與氧化壓力。

Obesity is one of the main public health problems in developed and developing countries. It serves as a significant risk factor for various diseases such as diabetes, cancer, heart disease, and hypertension. A previously study showed that the deep sea water (DSW) inhibits cell number of 3T3-L1 adipocytes and reduced body weight in animals. However, molecular mechanism of DSW on inhibition of adipogenesis is unclear. Therefore, the aim of this study was investigate the effect of DSW on adipogenesis in cell and animal models and its molecular mechanism. There are two topics included in this study, (1) Effect of DSW on inhibition of adipogenesis in 3T3-L1 adipocytes and its molecular mechanism. (2) Antiobesity effect of DSW on obesity rat induced by high-fat diet (HFD) and its molecular mechanism. (1) In cell culture model, the data indicated that the DSW does not inhibit cell numbers of 3T3-L1 pre-adipocytes (MTT assay) and adipocytes (oil o red staining) (p>0.05). But, DSW significantly decreased the intracellular triglyceride and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes. In gene expression, DSW also inhibits expressions of adipocyte differentiation (PPARγ, SREBP-1c, C/EBPα, and C/EBPβ), lipogenesis (ACC, SREBP-1a, and HMG-CoA reductase), and adipocytokine (leptin, IL-6, MCP-1, PAI-1, and CRP), and then up-regulated gene expressions of lipolysis and fatty acid oxidation (ATGL, HSL, CPT-1, and ACO). Our data indicated that DSW inhibits intracellular triglyceride and GPDH activity in 3T3-L1 adipocytes through modulation of gene expressions of adipogenesis and lipolysis. (2) In animal model, the results showed that the body weight [DSW group (1X-5X)], perirenal adipose tissue [DSW group (1X-5X)], liver organ [DSW group (2X-5X)] and, epididymal adipose tissue [DSW group (3X-5X)] in the DSW+HFD groups were significantly decreased as compared to the HFD group (p<0.05). The levels of hepatic triglyceride [DSW group (2X-5X)] and cholesterol [DSW group (3X-5X)] in the DSW+HFD groups were significantly decreased as compared to the HFD group (p<0.05). Serum triglyceride, insulin, and ALT [DSW group (1X-5X)] in the DSW+HFD groups were significantly decreased as compared to the HFD group (p<0.05). Serum glucose [DSW group (2X-5X)], cholesterol [DSW group (1X, 2X, and 5X)], free fatty acid [DSW group (1X, 3X, and 5X)], and AST [DSW group (5X)] in the DSW+HFD groups were significantly decreased as compared to the HFD group (p<0.05). The DSW+HFD groups showed microvesicular fat accumulation in the liver [DSW group (3X-5X)] and adipose tissue [DSW group (1X-5X)]. Moreover, fecal lipid [DSW group (1X-5X)], triglyceride [DSW group (2X-5X)], and cholesterol [DSW group (2X-5X)] output in the DSW+HFD groups were significantly increased as compared to the HFD group (p<0.05). Hepatic malondialdehyde (MDA) in DSW+HFD groups [DSW group (1X and 3X)] were significantly decreased as compared to the HFD group (p<0.05). Hepatic glutathione reductase (GRd) [DSW group (3X-5X)] in DSW+HFD groups were significantly increased as compared to the HFD group(p<0.05). In gene expression, DSW was significantly increased gene expressions of PPARα, AMPK, ACO, and CPT-1 in the hepatic tissue of rats with HFD-induced obesity (p<0.05). Moreover, DSW was significantly increased the gene expressions of lipolysis and fatty acid oxidation (ATGL, HSL, CPT-1, and ACO), and decreased the gene expressions of adipocytokine (TNF-α, PAI-1, and resistin) in adipose tissue of rats with HFD-induced obesity (p<0.05). These results demonstrate that intake of DSW can be beneficial for the suppression of high fat diet-induced dyslipidemia, hepatosteatosis, and oxidative stress in rats.

中文摘要 I
英文摘要 III
縮寫表 XII
前言 1
壹、文獻回顧 4
一、肥胖 4
(一) 肥胖之簡介 4
(二) 肥胖與代謝症候群 5
(三) 肥胖與脂肪酸調控 6
(四) 脂肪細胞所分泌之細胞激素 7
二、深層海水 16
(一) 深層海水簡介 16
(二) 深層海水研究現況 16
1. 改善皮膚過敏之研究 17
2. 脂質代謝調控之研究 17
3. 心血管疾病之研究 18
4. 氧化壓力之研究 21
5. 肥胖之研究 21
6. 腸道之研究 22
7. 骨質疏鬆之研究 22
8. 安全性研究 23
9. 其它生理相關之研究 23
(三) 深層海水之組成分 24
1. 鎂離子 24
2. 鈣離子 29
3. 鉀離子 32
(1) 鉀與血壓之研究 32
(2) 鉀與心血管與腦中風之研究 33
(3) 鉀與腎臟疾病之研究 33
(4) 鉀與骨質疏鬆之研究 34
貳、研究動機 35
参、材料方法 36
一、實驗設計 36
(一) 以體外細胞試驗模式釐清深層海水之抗肥胖作用與分子機轉 36
(二) 以體內動物試驗模式探討深層海水抑制高脂飲食誘導肥胖大鼠之影響及其分子機轉 37
二、材料 38
(一) 儀器 38
(二) 藥品 38
三、方法 40
第一部分-以體外細胞試驗模式釐清深層海水之抗肥胖作用與分子機轉 40
ㄧ、樣品 40
二、3T3-L1前脂肪細胞 40
三、3T3-L1前脂肪細胞之培養 40
四、脂肪細胞之分化培養 41
五、脂肪細胞Oil Red O染色 41
六、脂肪細胞胞內三酸甘油酯含量測定 42
七、脂肪細胞胞內glycerol-3-phosphate dehydrogenase (GPDH) 酵素活性分析 42
八、基因表現分析 42
(一) 細胞RNA萃取 42
(二) RNA濃度測定 43
(三) 反轉錄反應 (Reverse transcription) 43
(四) 即時定量反轉錄聚合酶連鎖反應 (Real time RT-PCR) 43
九、統計分析 44
第二部分-以體內動物試驗模式探討深層海水抑制高脂飲食誘導肥胖大鼠之影響及其分子機轉 45
ㄧ、動物、飲食與實驗設計 45
二、血清生化分析 46
三、肝臟總脂質分析 46
(一) 總脂肪萃取 46
(二) 三酸甘油酯測定 47
(三) 膽固醇測定 47
四、糞便總脂質分析 47
五、肝臟組織切片 47
六、抗氧化酵素分析 48
(一) 肝組織均質液製備 48
(二) 蛋白質含量測定 48
(三) 肝組織麩胱甘肽還原酶 (Glutathione reductase, GRd) 活性測定 48
(四) 肝組織麩胺基硫轉移酵素 (Glutathione S-transferase, GST) 活性測定 48
(五) 肝組織脂質過氧化物丙二醛 (malondialdehyde, MDA) 分析 49
(六) 肝組織總抗氧化能力測定 (trolox equivalent antioxidant capacity, TEAC) 49
(七) 血清總抗氧化能力測定 (trolox equivalent antioxidant capacity, TEAC) 49
七、基因表現分析 50
(一) 組織RNA萃取 50
(二) RNA濃度測定法 50
(三) 反轉錄反應 (Reverse transcription) 50
(四) 即時定量反轉錄聚合酶連鎖反應 (Real time RT-PCR) 50
八、統計分析 50
肆、結果 55
第一部分-以體外細胞試驗模式釐清深層海水之抗肥胖作用與分子機轉 55
一、深層海水對3T3-L1前脂肪細胞之細胞存活率之影響 55
二、深層海水對3T3-L1脂肪細胞之oil red O-stained material之影響 55
三、深層海水對3T3-L1脂肪細胞之胞內三酸甘油酯含量之影響 56
四、深層海水對調控3T3-L1脂肪細胞之胞內GPDH酵素活性之影響 56
五、深層海水對調控3T3-L1脂肪細胞相關訊息分子之影響 56
第二部分-以體內動物試驗模式探討深層海水抑制高脂飲食誘導肥胖大鼠之影響及其分子機轉 58
一、深層海水對高脂飲食誘導肥胖大鼠之體重、食物攝取量與能量攝取之影響 58
二、深層海水對高脂飲食誘導肥胖大鼠之臟器重量之影響 58
三、深層海水對高脂飲食誘導肥胖大鼠之血清生化參數之影響 59
四、深層海水對高脂飲食誘導肥胖大鼠之肝臟總脂質、三酸甘油酯與膽固醇含量之影響 59
五、深層海水對高脂飲食誘導肥胖大鼠之糞便總脂質、三酸甘油酯與膽固醇含量之影響 59
六、深層海水對高脂飲食誘導肥胖大鼠之肝臟與脂肪細胞組織切片之影響 60
七、深層海水對高脂飲食誘導肥胖大鼠之肝臟與血清抗氧化酵素之影響 60
八、深層海水對高脂飲食誘導肥胖大鼠之脂肪組織相關訊息分子之影響 60
九、深層海水對高脂飲食誘導肥胖大鼠之肝臟組織相關訊息分子之影響 61
十、深層海水對高脂飲食誘導肥胖大鼠之肝臟鎂離子濃度之影響 61
伍、討論 62
陸、總結論 74
柒、實驗圖表 75
捌、參考文獻 102

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行政院衛生署http://www.doh.gov.tw/CHT2006/index_populace.aspx
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