臺灣博碩士論文加值系統

　　男性和停經後女性在高血壓和冠狀動脈疾病的發生率比停經前女性高。一氧化氮 (NO)是一種血管內皮釋放的血管擴張劑，而雌激素能藉由增加NO的合成達到心血管保護效果。我們知道雌激素能藉由調節NO以增加環鳥糞嘌呤核苷單磷酸鹽(cGMP)達到血管平滑肌鬆弛的效果，但性別與補充雌激素對於NO-cGMP訊息下游的主要分子蛋白激酶G (PKG)的影響相關資料卻極少。我們研究的目標是探討性別對於PKG調節血管舒張的效果，我們在Sprague-Dawley大白鼠胸主動脈加入8-bromoguanosine-3',5'-cyclic monophosphate、zaprinast或BAY 41-2272影響PKG活性，並觀察血管放鬆的性別差異表現，結果顯示三種藥物對公鼠胸主動脈引起的血管鬆弛反應比體重對照母鼠較大。我們進一步觀察性激素對於PKG的調節，對於去除卵巢母鼠或去除睪丸公鼠進行性激素或空白試劑的補充，並利用定量聚合酶鏈鎖反應測量PKG的mRNA相對表現量。結果顯示對於去除卵巢母鼠補充雌二醇會造成主動脈PKG表現量下降，而黃體素能拮抗雌二醇所造成的反應；睪固酮同樣也會造成主動脈PKG表現量減少。我們的實驗結果顯示：(1) PKG所調節的血管舒張反應雄性高於雌性；(2) 性激素能調節主動脈的PKG表現。

The incidence of hypertension and coronary artery disease in men and postmenopausal women are higher compared with premenopausal women. Nitric oxide (NO) is a major vasodilator released by vascular endothelium and estrogen augments NO and cyclic guanosine monophosphate (cGMP) synthesis which may contribute to the observed cardioprotective effects. However, the effects of gender and estrogen administration on the primary downstream effector molecule of the NO-cGMP signaling pathway, protein kinase G (PKG) is not clear. The aim of our study was to characterize effects of gender and hormones on vasodilation mediated by PKG. The study of sexual dimorphism in PKG activity was based on relaxation of thoracic aortic segments of Sprague-Dawley rats by 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP), zaprinast and BAY 41-2272. All these reagents were more potent in aortas from male rats than those from weight-matched female ones. To further investigate the effects of sex hormones on PKG, ovariectomized (OVX) or orchiectomized (ORX) rats were supplied with sex hormones or vehicle. Relative mRNA expression of PKG was assessed by quantitative real-time PCR. Administration of 17β-estradiol to OVX rats decreased aortic PKG expression, and progesterone attenuated this response. Testosterone treatment of ORX rats also decreased aortic PKG expression. We thus conclude that 1) gender difference exists in PKG-mediated vasodilation, with males appear to be better and 2) sex hormones might modulate PKG expression in aorta.

目錄

指導教授推薦書
口試委員會審定書
國家圖書館授權書
長庚大學授權書
誌謝................................................................................................................................v
中文摘要......................................................................................................................vii
英文摘要……………………………………………………………………….……viii
縮寫表…………………………………………………………………………...……ix
目錄……………………………………………………………………………...……xi
圖表目錄…………………………………………………………………………….xiv
第一章 序論………………………………………………………………………...1
1.1 雄性素與雄性素對心血管之影響……………………………………………1
1.2 雌激素與雌性素對心血管之影響………………………………....................2
1.3 黃體內分泌與黃體內分泌對心血管之影響…………………………………4
1.4 一氧化氮－環鳥糞嘌呤核苷單磷酸鹽途徑…………………………………6
1.5 一氧化氮－環鳥糞嘌呤核苷單磷酸鹽途徑擴張血管的性別差異及雌激素之影響………………………………………………………………………...7
1.6 蛋白激酶G…………………………………………………………................8
第二章 研究目的與假說…………………………………………………………..10
2.1 研究目的…………………………………………………………...................10
2.2 假說……………………………………………………………………….…..10
第三章 實驗方法與材料…………………………………………………………..11
3.1 實驗材料…………………………………………………………...................11
3.1.1 實驗動物來源………………………………………………………..…..11
3.1.2 實驗藥品……………………………………………………………..…..11
3.1.3 實驗儀器……………………………………………………………..…..12
3.1.4 實驗試劑套組…………………………………………………………....13
3.2 實驗方法……………………………………………………………………...13
3.2.1 胸主動脈血管環張力反應試驗………………………………………....13
3.2.1.1 實驗分組………………………………………………………….....13
3.2.1.2 判定母鼠的動情週期…………………………………………….....14
3.2.1.3 胸主動脈血管環的製備………………………………………….....14
3.2.1.4 血管反應實驗前的血管活性測試……………………………….....15
3.2.1.5 ACh刺激的血管內皮依賴性之鬆弛反應……………………...…..16
3.2.1.6 BAY 41-2272刺激的血管鬆弛反應………………………………..16
3.2.1.7 Zaprinast刺激的血管鬆弛反應…………………………………......17
3.2.1.8 8-Br-cGMP刺激的血管鬆弛反應…………………………………...17
3.2.1.9 浸泡ODQ一段時間後，執行8-Br-cGMP刺激的血管鬆弛反應…..17
3.2.1.10 評估胸主動脈血管環張力反應……………………………….…...18
3.2.2定量聚合酶鏈鎖反應…………………………………………………..18
3.2.2.1 實驗分組………………………………………………………….….18
3.2.2.2 卵巢切除手術………………………………………….…………….20
3.2.2.3 睪丸去除手術……………………………………….…………….....20
3.2.2.4 三種性激素之配製與補充方法……………………….………….....21
3.2.2.5 定量聚合酶鏈鎖反應………………………………………….….....22
3.2.2.6 q-PCR實驗結果評估方法…………………………………………...23
3.3 統計分析……………………………………………………………….……...24
第四章 實驗結果…………………………………………………….……………..25
4.1 血管環的張力反應試驗………………………………….…………………...25
4.1.1 代表性實驗結果：ACh作用後血管環對ODQ與L-NNA之反應……..25
4.1.2 ACh造成之血管內皮層依賴性鬆弛反應………………………...….….25
4.1.3 8-Br-cGMP造成之血管鬆弛反應………………………………………..26
4.1.4 ODQ對8-Br-cGMP造成之血管鬆弛反應之作用……………………....26
4.1.5 於血管內皮層存在下，BAY 41-2272造成之血管舒張反應…………...27
4.1.6 血管內皮層去除後，BAY 41-2272造成之血管舒張反應……………...27
4.1.7 Zaprinast刺激的血管鬆弛反應……………………………………….….28
4.1.8 血管內皮層釋出的基礎NO效用……………………………….......29
4.2 eNOS與PKG-I的mRNA表現量………………………………………..…....29
4.2.1 血管張力實驗組eNOS的mRNA表現量…………………………….…29
4.2.2 血管張力實驗組PKG-I的mRNA表現量……………………………....30
4.2.3 雄性激素補充組PKG-I的mRNA表現量……………………………....30
4.2.4 雌性激素補充組PKG-I的mRNA表現量…………………………..…..31
第五章 討論………………………………………………………………………...48
5.1 ACh經由NO-cGMP途徑造成血管鬆弛反應存在性別差異………………..48
5.2 外源性8-Br-cGMP的造成血管鬆弛反應存在性別差異…………………...49
5.3 卵巢性荷爾蒙對血管內PKG-I的mRNA調節……………….……………..51
5.4 睪丸性荷爾蒙對血管內PKG-I的mRNA調節………………..…………….53
5.5 內源性cGMP亦造成PKG-I調控的血管鬆弛反應存在性別差異…………53
5.6 血管平滑肌內NO-cGMP途徑中其他存在性別差異之可能……………….54
5.7 比較年齡對照母鼠和體重對照母鼠兩種模型………………..……………..57
5.8 將胸主動脈與腹主動脈視為不同檢體進行研究…….……….……………..59
第六章 結論……………………………………………………………….………..61
參考文獻……………………………………………………………….……………..62
圖表目錄

圖一、以ACh進行內皮依賴性舒張反應的實驗結果圖形………………………....32
圖二、累積濃度ACh對SD大白鼠血管環的影響曲線圖…………………………33
圖三、累積濃度8-Br-cGMP對SD大白鼠血管環的影響曲線圖…………………34
圖四、待1 μM PE使收縮完全，給10 μM ODQ浸泡十五分鐘，再給予累積濃度
8-Br-cGMP，對SD大白鼠血管環的影響曲線圖………..………………...35
圖五、累積濃度BAY 41-2272對內皮完整的SD大白鼠血管環的影響曲線圖….36
圖六、累積濃度BAY 41-2272對內皮去除的SD大白鼠血管環的影響曲線圖…37
圖七、累積濃度zaprinast對SD大白鼠血管環的影響曲線圖…………………..…38
圖八、血管內皮層釋放之基礎NO效果比較圖……………………………………..39
圖九、在公母鼠胸主動脈eNOS的mRNA表現量………………………………….40
圖十、在公母鼠腹主動脈eNOS的mRNA表現量………………………………….41
圖十一、在公母鼠胸主動脈PKG-I的mRNA表現量………………………………42
圖十二、在公母鼠腹主動脈PKG-I的mRNA表現量………………………………43
圖十三、在雄性激素調節組胸主動脈PKG-I的mRNA表現量……………………44
圖十四、在雄性激素調節組腹主動脈PKG-I的mRNA表現量……………………45
圖十五、在雌性激素調節組胸主動脈PKG-I的mRNA表現量……………………46
圖十六、在雌性激素調節組腹主動脈PKG-I的mRNA表現量……………………47

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