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研究生:廖語彤
研究生(外文):Yu Tung Liao
論文名稱:年齡與自發性高血壓大鼠第二型一氧化氮合成酶表現的關係
論文名稱(外文):Age correlate with type II nitric oxide synthase expression in vascular smooth muscle cells of spontaneously hypertensive rats.
指導教授:趙壯飛呂美華呂美華引用關係陳正繹
指導教授(外文):Chung Faye ChaoMei Hua LuJang Yi Chen
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
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:52
中文關鍵詞:年齡一氧化氮合成酶自發性高血壓鼠
外文關鍵詞:agenitric oxide synthasespontaneously hypertensive rats
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中文摘要
誘導型一氧化氮合成酶(Inducible nitric oxide synthase, iNOS)在平常情況下並不表現,但在細菌內毒素或細胞激素等刺激下,即可大量被表現,根據本實驗室之前所做的研究結果顯示,成年(24週)的Spontaneously hypertensive rats(SHR)大鼠iNOS蛋白質表現量明顯比同週齡正常血壓大鼠Wistar-Kyoto rats(WKY)高,而在年輕(4週)的SHR及WKY大鼠並未發現此現象,因此為了釐清在SHR大鼠中iNOS基因的過度表現是因為高血壓的持續發展或是因為年齡的增加所造成,因此本論文使用不同週齡SHR及WKY初代培養的主動脈平滑肌細胞為材料,首先萃取出蛋白質以西方點墨法來分析iNOS蛋白質的表現,並以構築好含不同大小的iNOS啟動子的質體(-3.2、-1.7、-0.5kb)來轉殖於不同週齡的SHR及WKY主動脈平滑肌細胞並評估基因的表現。結果顯示在16及24週SHR大鼠iNOS蛋白質表現量明顯比同週齡的WKY大鼠高,在細胞轉染的結果中無論是SHR或是WKY,皆在iNOS promoter -1.7kb~-0.5kb位置有IL-1反應區段。而值得注意的是,SHR在此區段(-1.7kb~-0.5kb)有血壓及iNOS表現的相關性變化,WKY則無此變化,而調控iNOS promoter的轉錄因子(NF-B),在血管平滑肌細胞中蛋白質總表現量,在8、16、24週的SHR表現皆明顯高於同週齡的WKY。因此我們的結果證實SHR 血管平滑肌細胞中iNOS的表現與血壓有正相關的關係,而血壓上升會刺激NF-B的活化,則可能是iNOS大量表現的原因。

英文摘要
The inducible nitric oxide synthases (iNOS or type II NOS) can be induced by cytokine or LPS stimulation in vascular smooth muscle cells (VSMC). In our previous studies, the iNOS expression by cytokine stimulation in VSMC of the SHR at age of 24 weeks was much higher than in age-matched WKY rats, but there was no significantly different expression between SHR and WKY rats at the age of 4 weeks old. In this study, we try to verify that whether the overexpression of iNOS in VSMC of SHR at the age of 24 weeks and the different expression of iNOS between SHR and WKY rats were due to aging process or hypertension development. Therefore, aortic SMC were isolated and cultivated from different ages of SHR and WKY rats. After 48 hours treatment of Interleukin-1(IL-1β), the total protein were extracted from the cultured VSMC and analyzed by western blot for iNOS expression. For evaluation of iNOS different expression regulation in VSMC between SHR and WKY rats, a -3.2kb rat iNOS promoter construct with luciferase and -1.7kb as well as -0.5kb deletion mutant constructs were transfected to the IL-1β treated cells.
Ours results showed that at 16 and 24 weeks, the protein expressions of iNOS in the VSMC of SHR were significantly higher than in WKY. The iNOS promoter at the region of -1.7~-0.5Kb was responsible for the induction of iNOS expression in VSMC from all age groups of SHR and WKY. Interesting, the region of -1.7~-0.5Kb in SHR showed the relationship between hypertension and iNOS expression but this change was not found in WKY. In addition, at 8,16 and 24 weeks, the activity and protein expression of NF-B in the aortic SMC of SHR were significantly higher than those in WKY. In conclusion, our results demonstrated that increase of protein and gene expression of iNOS occurred in SHR was not age dependent rather than hypertension related even. The mechanism of higher expression regulation of iNOS in aged SHR VSMC may be correlated with higher expression of NF-B.

目錄
目錄………………………………………………………………………Ⅰ
圖目錄……………………………………………………………………Ⅲ
表目錄……………………………………………....................................Ⅳ
中文摘要…………………………………………………………………Ⅴ
英文摘要…………………………………………………………………Ⅵ
第一章、 緒論……………………………………………….....1
第一節、 一氧化氮發現史回顧……………………………………1
第二節、 一氧化氮生化特性簡介…………………………………1
第三節、 第二型一氧化氮合成酶在心臟血管系統的角色……....4
第二章、 實驗材料與方法………………………………….....6
第一節、 實驗材料............................................................................6
第二節、 實驗方法............................................................................9
一、 大鼠胸主動脈平滑肌細胞的培養…………………........9
二、 主動脈平滑肌細胞繼代培養…………………………...10
三、 大鼠胸主動脈平滑肌細胞的純度鑑定…………….…..11
四、 大鼠胸主動脈平滑肌細胞經轉染iNOS promoter後螢
光強度分析……………………………………………...12
五、 西方墨點法………………………………………….......14
六、 統計分析…………………………………………….......16
第三章、 實驗結果……………………………………………17
第一節、 大鼠胸主動脈平滑肌細胞的培養……………..……….17
1. 形態上的觀察………………………………………………..17
2. 血管平滑肌細胞經anti-smooth muscle-α-actin
染色定性……………………………………………………...17
第二節、 經IL-1β處理後不同週齡的SHR及WKY大鼠胸主
動脈平滑肌細胞iNOS的蛋白質表現………………….18
第三節、 iNOS promoter在不同週齡SHR以及WKY胸主動脈
平滑肌細胞內的表現情形………………………………18
第四節、 iNOS的表現與高血壓的變化…………………………..20
第五節、 經IL-1β處理後不同週齡的SHR及WKY大鼠胸主動脈平滑肌細胞NF-B的蛋白質總量及活化態蛋白表現與血壓形成的變化關係…………………………………………………………..21
第四章、 討論………………………………………………….23
第五章、 實驗結果(圖表)………………………………….28
第六章、 附錄………………………………………………….40
第七章、 參考文獻……………………………………….........48
圖目錄
圖1. WKY大鼠血管平滑肌細胞………………………......................28
圖2. SHR大鼠血管平滑肌細胞………………………........................29
圖3. SHR大鼠血管平滑肌細胞純度鑑定……………………………30
圖4. WKY大鼠血管平滑肌細胞純度鑑定…………………………..31
圖5. WKY以及SHR 大鼠血管平滑肌細胞iNOS的蛋白質表現…32
圖6. SHR的iNOS promoter表現強度……………………………….34
圖7. WKY的iNOS promoter表現強度………………………………35
圖8. WKY以及SHR的iNOS promoter表現強度…………………..36
圖9. WKY以及SHR的iNOS promoter表現強度與血壓的變化…...37
圖10. WKY以及SHR 大鼠血管平滑肌細胞NF-B的蛋白質表現..38
圖11. WKY以及SHR 大鼠血管平滑肌細胞I-B-p的蛋白質表現...39
附錄目錄
附錄一、pGL3-basic vector示意圖……………………………………41
附錄二、pRL-CMV vector示意圖………………………………….....42
附錄三、Sequence of type II NOS promoter…………………………..46
附錄四、iNOS promoter不同大小片段示意圖………………………43
附錄五、不同週齡的WKY及SHR血壓表………………………....44
附錄六、iNOS promoter binding site示意圖………………………....45

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