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研究生:張力元
研究生(外文):Lee-Yuan Chang
論文名稱:丙硫氧嘧啶引發低甲狀腺機能鼠血脂質變異對心血管組織細胞內造骨蛋白基因表現的影響
論文名稱(外文):Effects of Hypercholesterolemia on Osteopontin Gene Expression in the Aorta and Heart of Hypothyroid Mice Induced by Propyl-thio-uracil
指導教授:劉英明
指導教授(外文):Ying-Ming Liou
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:86
中文關鍵詞:造骨蛋白丙硫氧嘧啶動脈粥狀硬化甲狀腺素低能症
外文關鍵詞:osteopontinpropylthiouracilatherosclerosishypothyroidism
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造骨蛋白 (Osteopontin, OPN) 為細胞外基質蛋白,當心肌梗塞與動脈粥狀硬化發生,心血管組織內OPN就會大量表現。利用實驗動物─Apo E脂蛋白基因剔除鼠,在12與22週齡,觀測到動脈血管組織OPN mRNA有明顯增加;而在22週齡,心臟組織內也會增加OPN mRNA的表現。顯示Apo E脂蛋白基因剔除鼠之血脂代謝異常可能經由增加細胞內OPN基因的表現,因而造成血管組織的病變,最後危害心血管功能。本研究利用另一種實驗動物模式─餵食抗甲狀腺激素藥物 (丙硫氧嘧啶,PTU) 引起低甲狀腺機能鼠以及增加實驗動物血脂濃度─來測試對心臟與動脈血管組織內OPN mRNA以及蛋白表現的影響。利用半定量反轉錄酶聚合酶連鎖反應、酵素免疫分析法以及西方轉漬法等實驗方法分別來分析組織細胞內mRNA以及蛋白的表現。實驗結果如下:1.低甲狀腺機能鼠的動脈組織,在4~10週,表現的OPN mRNA較正常動物高出1.4倍,而在22週後,則高出2倍多;心臟組織表現的OPN mRNA,只有在22週後才有明顯增加 (~1.5倍) ;2.與mRNA表現的實驗結果相符;血管組織OPN蛋白表現量增加1.5倍 (10週) ;心臟組織增加~1.7倍 (22週) 。3.OPN蛋白在血管組織表現的分子大小有~66 kD以及~50 kD;心臟組織則以~50 kD為主。低甲狀腺機能鼠的動脈組織,有減少~66 kD 以及增加~50 kD的蛋白表現,而心臟組織表現的蛋白形式沒有改變。這些研究結果顯示:低甲狀腺機能鼠血脂濃度增加會造成心血管組織OPN mRNA以及蛋白表現增加。因此,OPN基因在心血管組織表現或許可以被用來當作偵測心血管疾病的分子標記。
It has been shown that osteopontin (OPN), an extracellular matrix protein, is highly expressed in both cardiac and vascular tissues of the animals afflicted with cardiovascular disease. Using the atherosclerotic model of Apo E gene knock out mice, we have found that malfunction of cholesterol metabolism would lead to the increases in the levels of OPN mRNA expression in the aorta of mutated animals at 12- and 22- weeks, while in the heart of 22 week old . To further test if the disturbance by hypercholesterolemia would cause the increases in the OPN gene expression in cardiovascular tissue, we used another animal model of hypothyroid mice induced by anti-thyroid drug, propylthiouracil (PTU). After 2 weeks of drinking PTU-containing water, the animals have significant decreases in thyroid hormones (T3 and T4) and immediate increases in the cholesterol levels. Semi-quantitative RT-PCR analysis showed that OPN mRNA expression in the arota of hypothyroid mice has increased by 1.4-fold at 4 week and 2-fold at 22 week, while the levels of the heart of hypothyroid mice increased by1.5-fold at 22 week. In consistence with the mRNA expression, the OPN protein expression levels measured with ELISA analyses showed ~1.5- and ~1.7-fold increases in the aorta (10 weeks) and the heart (22 weeks) of hypothyroid mice, respectively. There are two different isoform of OPN (66-kD and 50-kD) expressed in the aorta of mice. However, the expression pattern is different in the aorta of control and hypothyroid mice. The expression of 66-kDa OPN is decreased but increased by the presence of 50-kDa OPN in the hypothyroid mice. In contrast, only one major protein of 50-kDa OPN is present in the heart of control and hypothyroid mice. All together, the data obtained indicated that the hypothyroid mice with hypercholesterolemia induced by PTU could increase OPN mRNA and protein expression in the aorta and the heart. The findings from this study might suggest that the increase in OPN gene expression in the aorta and heart could serve as a molecular marker for cardiovascular diseases.
中文摘要....................................................1
英文摘要....................................................3
第一章、前言................................................5
第二章、材料與方法..........................................25
第三章、結果................................................42 第四章、討論................................................63
第五章、參考文獻............................................76
圖 目 錄
附圖一、動脈粥狀硬化之過程...................................16
附圖二、OPN的分子結構.......................................22
附圖三、OPN的生物功能.......................................23
附圖四、Propylthiouracil (PTU) 的化學結構....................26
附圖五、動脈之插管...........................................28
附圖六、動脈插管與液態壓力轉換器相接情形.....................28
附圖七、多用途記錄器的血壓記錄圖.............................29
圖一、分析不同週齡Apo E 基因剔除鼠之主動脈與
心臟組織OPN mRNA表現差異..............................43
圖二、正常與PTU誘導的老鼠血清T4與T3含量之影響.............45
圖三、正常與PTU誘導的老鼠血清
HDL-C/TC與LDL-C/TC之比例.............................50
圖四、正常與PTU誘導的老鼠血管組織
OPN mRNA表現量之差異..................................55
圖五、正常與PTU誘導的老鼠心臟組織
OPN mRNA表現量之差異..................................56
圖六、ELISA分析已知濃度OPN standards的OD值
以及繪製的OD─蛋白濃度相關曲線........................58
圖七、正常與PTU誘導的老鼠 (10週) 血管組織
OPN蛋白濃度之差異....................................59
圖八、正常與PTU誘導的老鼠 (4週、10週、22週)
心臟組織OPN蛋白濃度之差異............................60
圖九、西方轉漬法分析心臟、血管與腎臟三種組織
細胞內OPN蛋白表現的分子量............................62
表 目 錄
表一、核酸引子...............................................35
表二、正常與PTU誘導的老鼠之體重.............................46
表三、正常與PTU誘導的老鼠之血脂値...........................49
表四、正常與PTU誘導的老鼠之心跳速率.........................52
表五、正常與PTU誘導的老鼠之血壓.............................53
表六、Apo E基因剔除鼠與低甲狀腺機能鼠之比較.................74
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