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研究生:陳聖壬
研究生(外文):Shen-Jen Chen
論文名稱:高膽固醇餵食對大鼠主動脈內皮細胞的影響
論文名稱(外文):Effects of High-cholesterol Feeding on Endothelial Cells in Intact Rat Aortas
指導教授:任卓穎
指導教授(外文):Chau-Ying Jen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:鈣離子動脈硬化內皮細胞
外文關鍵詞:atherosclerosiscalciumendothelial cells
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在文明世界中,動脈硬化是罹病率和致死率的主要因素之一。在動脈硬化的發展過程中,動脈內皮細胞的損害,已是長久以來被認為為最早發生的現象。而單核球沾黏到主動脈內皮細胞的表面進而游走進入內皮細胞是一個最早可以偵測到的細胞級現象。血管內皮細胞在調控許多重要的生理功能上扮演一個相當重要的角色,諸如血管新生、凝血、免疫反應以及血管張力,而在內皮細胞中,細胞內鈣離子是一個極為重要的訊息傳遞者。至目前為止的研究並無直接證據指出初期動脈硬化與內皮細胞細胞內鈣離子濃度上升的改變有關。欲探討在初期動脈硬化中,內皮細胞內鈣離子訊息是否扮演一個重要的角色,本研究選擇9(餵食高膽固醇飼料3週) 、12(餵食6週) 及15(餵食9週) 週齡Wistar大鼠作為實驗模式,而以餵食正常飼料的大鼠作為對照組,取其胸主動脈後,給予乙醯膽鹼(ACh) 及組織胺(His) 刺激內皮細胞後,利用本實驗室所發展的技術測得內皮細胞細胞內鈣離子濃度上升的變化。結果發現:(1) 相同週齡的實驗組大鼠體重明顯比控制組大鼠要低;(2) 實驗組大鼠血清中脂肪含量皆比控制組高,但會隨週齡增加而逐漸下降;(3) 於實驗組明顯發現白血球沾黏於胸主動脈內皮細胞上;(4) 控制組大鼠對於刺激劑所誘發的鈣離子濃度上升普遍比實驗組大鼠高;(5) 內皮細胞細胞內之鈣離子濃度基礎值以15週齡實驗組最高。總結,在9週齡實驗組大鼠,其血清脂肪成分含量最高,白血球沾黏的數量並不多,但明顯的內皮細胞細胞內鈣離子反應變差;而隨著週齡的增加,血清中脂肪成分含量逐漸下降,但白血球沾黏的數量卻逐漸增加,而內皮細胞細胞內鈣離子反應也逐漸回復。
Atherosclerosis is a leading cause of mortality and morbidity in the civilized world. Endothelial injuries in the artery have long been considered as initial events in the pathogenesis of atherosclerosis. In experimental animals fed an atherogenic diet, adhesion of monocytes as well as T lymphocytes to the arterial endothelial surface, and sequential migration into the subendothelial space is one of the earliest detectable cellular events. Vascular endothelium plays an important role in regulating many important physiological functions, such as angiogenesis, hemostasis, immune defense, and vascular tone. Intracellular free calcium ion, [Ca2+]i, is one of the most common signal transduction elements in the endothelial cells (ECs). However, the detailed relationship of early-stage atherosclerosis and the change of calcium elevation in endothelial cells is poorly understood. To investigate whether alterations in the endothelial [Ca2+]i signaling can serve as an important marker in the early-stage atherosclerosis, we used our newly developed technique to monitor endothelial [Ca2+]i signaling stimulated by acetylcholine (ACh) and histamine (His) in thoracic aortas of Wistar rats fed with normal or high-cholesterol diets. The experimental groups were fed the high-cholesterol diet (3 weeks for the 9-wk-old group, 6 weeks for the 12-wk-old group, 9 weeks for the 15-wk-old group), whereas the control groups were fed the normal diet. Our results showed that 1) the body weights of the high-cholesterol diet-fed rats were lower than those of the control rats through the entire period of the experiment; 2) once elevated, the total plasma cholesterol level in the experimental groups decreased with the feeding duration of high cholesterol diet; 3) adherence of mononuclear cells markly increased in the experimental groups; 4) agonist-induced endothelial [Ca2+]i elevation in the control groups were higher than the experimental groups; 5) when compared with that in 6-wk-old rats, the basal endothelial [Ca2+]i in 15-wk-old experimental rats were higher. In summary, high cholesterol diet feeding in Wistar rats increased their total plasma cholesterol level, induced adherence of mononuclear cells to aortic endothelium surrounding the intercostal orifices, and markly reduced the agonist-induced endothelial [Ca2+]i elevation. Although both level of total plasma cholesterol and the agonist-induced endothelial [Ca2+]i elevation gradually recovered, the adherence of mononuclear cells progressively increased with the duration of high cholesterol diet feeding.
緒論.............................................1
材料與方法.......................................7
一、材料.........................................7
(一)實驗動物.....................................7
(二)藥品.........................................7
(三)其他.........................................9
二、儀器設備....................................10
三、實驗設計....................................11
(一)大鼠飼料餵食設計............................11
(二)測量參數....................................12
1.測量大鼠的體重及進食變化...................12
2.測量大鼠血清中脂肪成份的含量...............12
3.用掃瞄式電子顯微鏡觀察白血球沾黏...........12
4.Oil red O染色以觀察血管壁脂肪堆積的情形....13
5.分別測定並比較不同週齡週控制組及實驗組大鼠之胸主動脈在給予ACh以及His刺激後所誘發內皮細胞細胞內鈣離子濃度變化.......................13
四、方法........................................14
(一)溶液的製備..................................14
(二)血管段的製備................................15
(三)掃瞄式電子顯微鏡樣本的製備..................15
(四)Oil red O染色的樣本製備.....................17
(五)fura 2的染色 ( Fura 2 Loading ).............18
(六)組織流體室的組裝及血管的固定................18
(七)儀器的裝置及實驗進行步驟....................19
(八)測量由ACh及His所誘發之內皮細胞細胞內鈣離子濃度變化........................................20
(九)內皮細胞內鈣離子濃度的測定與校正............21
(十)統計分析....................................22
結果............................................23
一、控制組及實驗組大鼠的體重及進食變化情形......23
二、分別測量並比較不同週齡控制組及實驗組大鼠血清中脂肪成份的含量..................................23
三、分別觀察並比較不同週齡控制組及實驗組大鼠之沾黏於胸主動脈內皮細胞上的白血球其分佈情形及數量....25
四、分別觀察並比較不同週齡控制組及實驗組大鼠之胸主動脈在oil red O染色後血管內壁上脂肪堆積情形.....27
五、測量及比較不同週齡控制組及實驗組大鼠之胸主動脈在給予ACh以及His刺激後所誘發內皮細胞細胞內鈣離子濃度變化情形......................................28
(一)不同週齡控制組及實驗組之內皮細胞細胞內鈣離子濃度上升曲線及影像................................28
(二)6週齡控制組不同區域之內皮細胞細胞內鈣離子濃度上升之比較......................................29
(三)9週齡控制組及實驗組之比較...................30
(四)12週齡控制組及實驗組之比較..................31
(五)15週齡控制組及實驗組之比較..................32
(六)不同週齡控制組對於ACh反應之比較.............33
(七)不同週齡實驗組對於ACh反應之比較.............34
(八)不同週齡控制組對於His反應之比較.............35
(九)不同週齡實驗組對於His反應之比較.............36
(十)不同週齡控制組及實驗組大鼠之基礎內皮細胞細胞內鈣離子濃度變化..................................37
討論............................................38
結論............................................44
參考文獻........................................46
圖..............................................55
附圖............................................80
附錄............................................83
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