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研究生: 賴佩郁
Pey-Yuh Lai
論文名稱: 大豆異黃酮對肝細胞、腎間質細胞脂質堆積與發炎,及停經婦女之影響
The Effects of Soy Isoflavone on Lipid accumulation and Inflammation in HepG2 cells, Mouse renal mesangial cells and Postmenopausal Women
指導教授: 吳文惠
Wu, Wen-Huey
學位類別: 碩士
Master
系所名稱: 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 109
中文關鍵詞: 大豆異黃酮停經婦女脂質堆積HepG2小鼠腎間質細胞
英文關鍵詞: soy isoflavones, postmenopausal women, lipid accumulation, HepG2, mouse mesangial cell
論文種類: 學術論文
相關次數: 點閱:293下載:10
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  • 停經婦女由於缺乏雌激素,會造成體重、腹部脂肪量、腰圍以及心臟病危險因子增加。許多研究指出肥胖與非酒精性脂肪肝或是腎臟脂毒性之間具有高度相關。大豆異黃酮為植物性雌激素之一種,對心血管疾病與代謝症候群具有保護作用。本研究分別以人體及細胞實驗模式,探討介質研磨豆漿對於停經婦女之影響,以及大豆異黃酮對肝細胞、腎間質細胞脂質堆積與發炎之影響。
    人體實驗:招募10位健康、腰圍大於80 cm、停經一年以上之婦女,採用隨機、單盲之交叉試驗。試驗期5人先飲用介質研磨豆漿4週,經3週的洗滌期 (wash-out period)後,改飲用傳統豆漿4週,另外5人次序相反,每日分早晚兩次飲用 (500 ml/day)。比較體重、BMI、腰圍、腰臀比、血糖、胰島素、creatinine、GOT、GPT、血脂 (TG、TC、NEFA、HDL、LDL)、發炎因子CRP與性激素結合球蛋白(SHBG)濃度的變化。結果顯示上述所有數值不因投予傳統豆漿或介質研磨豆漿而有顯著改變。另分析受試者之尿液異黃酮含量,結果發現攝取介質研磨豆漿而非傳統豆漿,有顯著增加尿中異黃酮含量。
    細胞實驗:以人類肝癌細胞株 (HepG2)為模式,以1 mM脂肪酸 (Oleic acid、Palmitic acid、Stearic acid、Linoleic acid、Arachidonic acid, 1 mM FA)或5 μl/ml intralipid兩種模式誘發細胞內TG堆積,並投予不同劑量之大豆異黃酮 (daidzein, genistein與equol),共同培養24小時或48小時。結果發現,脂肪酸模式下,大豆異黃酮對肝細胞內TG與膽固醇含量無顯著影響;但在intralipid模式,大豆異黃酮能顯著降低肝細胞內TG與膽固醇含量。
    細胞實驗:以小鼠腎間質細胞 (mouse renal mesangial cell, MC)為模式,以1 mM FA誘發細胞內TG堆積,並投予不同劑量之大豆異黃酮共同培養24小時。結果顯示,大豆異黃酮對MC細胞內TG含量無顯著影響,但發現equol能顯著下降細胞內膽固醇含量。另外,用此刺激模式誘發MC細胞分泌促發炎激素單核球趨化蛋白-1 (monocyte chemoattractant protein-1, MCP-1),結果發現daidzein、genistein、equol皆能顯著下降MC細胞分泌MCP-1。
    總結:介質研磨豆漿或許能提高停經婦女對大豆異黃酮的生物利用率。在體外實驗,大豆異黃酮能降低intralipid誘發之肝細胞TG與膽固醇含量,並且能顯著降低由FA誘發之腎間質細胞分泌MCP-1。

    Postmenopausal women increase body weight, abdominal fat mass, waist circumference, and risks of heart disease due to the lack of estrogen. Many studies revealed a strong association between obesity and nonalcoholic fatty liver, or renal lipotoxicity. Soy isoflavones, partly as a phytoestrogen, have protective effects on cardiovascular diseases and metabolic syndrome. This study investigated the effects of media-milled soymilk in postmenopausal women and soy isoflavones on lipid accumulation and inflammation in HepG2 cells and mouse renal mesangial cells (MC).
    In human study, a randomized, single-blinded and crossover-controlled trial was conducted on ten apparently healthy postmenopausal women with the waist circumference over 80 cm. Subjects were supplemented with 500 ml/day either media-milled soymilk or tranditional soymilk for 4 weeks each, which were intervened by a wash-out period of 3 weeks. Fasting blood and first morning urine samples were collected at baseline and at the end of each feeding phase for the measurements of blood biochemical indices (glucose, insulin, creatinine, GOT and GPT), blood lipids (TG, TC, NEFA, HDL and LDL), C-reactive protein and sex-hormone binding globulin. No significant changes in these parameters were found. Analysis of urinary genistein, daidzein, and equol showed the levels were increased significantly by media-milled soymilk but not traditional soy milk.
    In HepG2 study, cells were treated with 1 mM fatty acid (FA) mixture containing oleic, palmitic, stearic, linoleic and arachidonic acids, or 5 μl/ml intralipid for 24h or 48h to induce intracellular TG accumulation. The results showed the co-incubation of daidzein, genistein or equol did not decrease intracellular TG and cholesterol content in 1 mM FA model, but significantly suppressed intracellular TG and cholesterol accumulation in intralipid model.
    In MC study, cells were treated with 1 mM FA mixture with or without daidzein, genistein and equol for 24h to induce intracellular TG and cholesterol accumulation. Isoflavones did not affect intracellular TG content, but intracellular cholesterol level was decreased signigicantly by equol. Secretion of monocyte chemoattractant protein-1(MCP-1) was increased by FA, but the level was significantly suppressed by the co-incubation of genistein, daidzein or equol.
    In conclusion, media-milled soymilk may increase the bioavailability of isoflavones in postmenopausal women. Genistein, daidzein and equol decrease intralipid-induced TG and cholesterol accumulation in HepG2, and significantly suppressed FA-induced MCP-1 secretion in MC cells.

    中文摘要.....I Abstract....III 目錄....V 表目錄...IX 圖目錄...X 第一章 緒論....... 1 第二章 文獻探討....3 第一節 大豆異黃酮 (Soy isoflavone)..3 壹、大豆之介紹.....3 貳、大豆異黃酮之種類及代謝... 4 参、大豆異黃酮之生理活性.....8 第二節 停經婦女 (Postmenopausal women)..... 12 壹、停經婦女之定義..12 貳、停經婦女之生理變化...... 12 参、停經婦女與腹部肥胖及代謝症候群之關係........13 肆、腹部肥胖造成之可能病理機轉........14 第三節 奈米科技 (Nanotechnology)...16 壹、奈米之定義.....16 貳、奈米科技...... 17 第四節 非酒精性脂肪肝病 (Non-alcoholic fatty liver disease, NAFLD)..20 壹、非酒精性脂肪肝病之定義與病理機轉......... 20 貳、大豆異黃酮對非酒精性脂肪肝之影響......... 21 第五節 腎臟損傷 (Renal injury)............ 22 壹、腎臟脂毒性 (Renal lipotoxicity)與發炎之影響.............22 貳、大豆異黃酮對腎臟損傷之影響........24 第三章 大豆異黃酮對停經婦女代謝症候群之影響..... 25 第一節 前言.............. 25 第二節 研究材料與方法.............. 26 壹、研究對象............ 26 貳、研究設計............ 26 參、研究材料與藥品..29 肆、樣本收集與處理..33 伍、分析項目與方法..34 第三節 結果.............. 37 壹、受試者基本資料........37 貳、受試者體重、腰臀圍與血壓之變化情形........37 参、受試者血糖與肝腎功能之變化情形...37 肆、受試者血脂、發炎因子及性激素結合球蛋白之變化情形.... 37 伍、受試者尿液異黃酮含量之變化情形...38 第四節 討論與結論..46 壹、受試者基本資料........46 貳、受試者體重、腰臀圍與血壓之變化情形........46 参、受試者血液生化值之變化情形......47 肆、受試者血脂及發炎因子之變化情形...47 伍、受試者性激素結合球蛋白之變化情形. 48 陸、受試者尿液異黃酮之變化情形......48 柒、結論........49 第四章 大豆異黃酮對肝細胞脂質堆積之影響.51 第一節 前言...... 51 第二節 研究材料與方法...... 52 壹、實驗設計.... 52 貳、研究材料.... 52 参、樣品製備及取得....... 53 肆、細胞存活率分析 (MTT assay).....54 伍、細胞內蛋白質濃度測定...55 陸、細胞內三酸甘油酯含量測定....... 56 柒、細胞內膽固醇含量測定...56 捌、統計分析.... 57 第三節 結果...... 58 壹、樣品對HepG2細胞存活率之影響.... 58 貳、樣品對HepG2細胞內三酸甘油酯含量之影響.... 58 参、樣品對HepG2細胞內膽固醇含量之影響....... 59 第四節 討論與結論..64 壹、脂肪酸與intralipid兩種刺激模式.64 貳、大豆異黃酮在兩種模式下對肝細胞脂質堆積之影響....... 65 参、結論........67 第五章 大豆異黃酮對腎間質細胞脂質堆積與發炎之影響.68 第一節 前言...... 68 第二節 研究材料與方法...... 69 壹、實驗設計.... 69 貳、研究材料.... 69 参、樣品製備及取得....... 70 肆、細胞存活率分析 (MTT assay)... 71 伍、細胞內蛋白質濃度測定...72 陸、細胞內三酸甘油酯含量測定........72 柒、細胞內膽固醇含量測定...72 捌、MCP-1蛋白質含量測定...72 玖、統計分析.... 73 第三節 結果...... 74 壹、樣品對小鼠腎間質細胞存活率之影響..74 貳、樣品對小鼠腎間質細胞內三酸甘油酯含量之影響..74 参、樣品對小鼠腎間質細胞內膽固醇含量之影響.... 74 肆、脂肪酸誘導小鼠腎間質細胞MCP-1分泌之時程.. 75 伍、樣品對脂肪酸誘導小鼠腎間質細胞分泌MCP-1之影響..... 75 第四節 討論與結論..81 壹、脂肪酸模式之建立......81 貳、大豆異黃酮對腎間質細胞毒性之影響. 81 参、大豆異黃酮對腎臟脂質堆積與發炎之影響......82 肆、結論....... 83 第六章 總結與未來建議....... 84 第七章 參考文獻....87 附錄一... 102 附錄二... 103 附錄三... 105 附錄四... 107 附錄五... 109

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