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系統識別號 U0026-0812200911425837
論文名稱(中文) 雌激素處理對apoE基因剔除母鼠活性氧分子的產生與動脈粥狀硬化進程之影響
論文名稱(英文) The effects of estrogen treatment on reactive oxygen species production and atherosclerosis progression in female apoE-/- mice
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 93
學期 2
出版年 94
研究生(中文) 陳亞琪
研究生(英文) Ya-Chi Chen
學號 t9692403
學位類別 碩士
語文別 中文
論文頁數 81頁
口試委員 指導教授-江美治
口試委員-陳洵瑛
口試委員-陳麗玉
中文關鍵字 雌激素  動脈粥狀硬化 
英文關鍵字 estrogen  atherosclerosis 
學科別分類
中文摘要 中文摘要
  心血管疾病是導致更年期後婦女死亡的主因,許多流行病學研究指出雌激素具有保護婦女罹患心血管疾病的作用,以動物模式進行的研究指出,雌激素與抑制動脈硬化的發展有關,但是由美國國家衛生研究院設計的兩個大型的臨床研究結果顯示,賀爾蒙替代療法對於更年期後婦女心血管疾病的影響目前還沒有定論。動脈粥狀硬化是冠狀動脈疾病及中風等心血管疾病是的重要機制,而影響動脈粥狀硬化發展的因子有很多。目前已有研究指出,血管中活性氧分子(reactive oxygen species)的產生是造成動脈粥狀硬化發展的一個很重要的因子。在動脈粥狀硬化的發展病程中,雌激素是否藉由抑制血管中活性氧分子的產生以抑制動脈粥狀硬化的發展,目前為止並不十分清楚。因此本實驗的目的在探討長期灌食雌激素對於抑制apoE 基因剔除母鼠動脈粥狀硬化的病程發展與活性氧分子產生的影響,並進一步探討雌激素的處理對生成與清除活性氧分子的酵素其表現量及活性的作用。

  本實驗採用去卵巢之apoE 基因剔除母鼠,共分為三組,分別為:一、低劑量雌激素實驗組,以灌食的方式每天給予1.25μg雌激素。 二、高劑量雌激素實驗組,以灌食的方式每天給予6.25μg雌激素。三、對照組,以灌食的方式每天給予5%酒精,三組動物皆餵食含有0.15%膽固醇但不含雌激素的高脂飼料,經過12週處理後將其犧牲。利用Oil red-O染色方法分析主動脈基部的動脈硬化斑區域,並進行影像量化分析。結果顯示低劑量與高劑量雌激素實驗組及對照組其動脈粥狀硬化區域分別為0.21±0.01, 0.20±0.01 與 0.32±0.02 mm2/section (n=6),實驗組較對照組顯著減少。此外,本實驗亦將主動脈基部切片以Hematoxylin &Eosin 染色並分析其內膜面積。結果顯示,低劑量與高劑量雌基素實驗組內膜層的面積(0.50±0.06, 0.44±0.02 mm2/section)均較對照組(0.69±0.04 mm2/section)明顯地減少。利用dihydroethidium染色的實驗探討血管中超氧自由基(O2•-)的產量,結果顯示以處理高劑量雌激素處理組,血管內膜層中超氧自由基的產量較對照組顯著地減少。血管中NADPH 氧化酶次單元 p22 phox的表現量在雌激素處理組明顯地減少,而另一個次單元 NOX4在各個處理組間則沒有明顯的差異。另外,將大白鼠平滑肌細胞同時處理雌激素(10-8M)及TNF-α(5ng/ml),對於TNF-α刺激NADPH 氧化酶活性的效果顯著地抑制。此外,在高劑量的雌激素處理組銅鋅過氧化物轉化酶及觸酶這兩種抗氧化酵素的表現量都顯著增加,而錳過氧化物轉化酶表現量的增加則未達到統計上的差異。雌激素的處理對於血管中平滑肌分化標記蛋白,包括SM-α-actin, SM-MHC, NM-MHC 及 h-caldesmon,的表現量並沒有顯著的改變。

  由本實驗的結果得知,以灌食的方式長期處理雌激素可減緩apoE基因剔除母鼠動脈粥狀硬化的病程發展。雌激素抗動脈粥狀硬化的作用中,可能藉由減少NADPH 氧化酶次單元的表現量及其活性並且增加抗氧化酵素的表現量來抑制活性氧分子的產生。
英文摘要 Abstract
 Cardiovascular diseases are among the leading causes of death in postmenopausal women. While the death rate from cardiovascular diseases are greater in men than in women during ages of 25-55 years , this difference in mortality rate disappears after menopause, suggesting that sex hormones provide protective effects on premenopausal women. Although the effects of hormone replacement therapy on cardiovascular diseases remain controversial, many studies reported that estrogen reduces cardiovascular diseases in animal models.

 Atherosclerosis is the underlying mechanism for cardiovascular diseases. Previous studies showed that oxidative stress, resulting from the imbalance between the production of reactive oxygen species (ROS) and the free radical scavenging enzymes, plays a pivotal role in the pathogenesis of atherosclerosis. Estrogen has been demonstrated to inhibit the production of ROS and increase free radical scavenging enzymes in cultured vascular smooth muscle cells and endothelial cells. Whether estrogen reduces ROS production in these cells in vivo remains unknown. Therefore, we hypothesized that estrogen inhibits atherosclerosis progression by decreasing oxidative stress in ovariectomized female apoE-/- mice. Twelve weeks-old ovariectomized female apoE-/- mice fed 0.15% cholesterol-containing diet were orally administered with estrogen (1.25 or 6.25μg /day) for 12 weeks. Our data showed that atherosclerotic lesion area in the aortic root detected by oil red-O staining was reduced by estrogen treatment (0.21±0.01, 0.20±0.01 and 0.32±0.02 mm2/section for 1.25μg/day, 6.25μg /day estrogen and control, respectively, n=6). Intima area in the aortic root detected by hematoxylin & eosin staining was also reduced by estrogen treatment (0.50±0.06, 0.44±0.02 and 0.69±0.04 mm2/section for 1.25μg /day, 6.25μg /day estrogen and control, respectively, n=6). Superoxide anion production in aortic sections detected by dihydroethidium staining markedly decreased in the estrogen (6.25μg/day)-treated group compared to the control. The expression of NADPH oxidase subunit p22 phox in thoracic aorta was decreased by estrogen treatment, while the expression of another subunit Nox4 was not affected. The NADPH oxidase activity stimulated by TNF-α (5ng/ml) was markedly diminished by estrogen (10-8M) in rat aorta smooth muscle cells. The expression of free radical scavenging enzymes, including Cu/Zn SOD and catalase, was significantly increased by estrogen (6.25μg/day) treatment whereas increases in Mn SOD did not reach statistical significance. The expression of smooth muscle differentiation markers, including SM-α-actin, SM-MHC, NM-MHC and high molecular weight caldesmon, was not affected by estrogen. These results demonstrate that long-term estrogen treatment by oral administration inhibits atherosclerosis progression in apoE-/- mice. Moreover, the atheroprotective effects of estrogen are at least in part mediated by its antioxidative actions both through increasing the expression of free radical scanvenging enzymes and through decreasing the expression and activity of NADPH oxidase.
論文目次 目錄
圖表目錄--------------------------------------------------1
緒論--------------------------------------------------------3
研究動機-------------------------------------------------14
實驗目的------------------------------------------------ 15
材料與方法--------------------------------------------- 16
藥品儀器-------------------------------------------------33
結果------------------------------------------------------ 38
討論------------------------------------------------------ 45
圖表-------------------------------------------------------51
參考文獻------------------------------------------------ 68
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