臺灣博碩士論文加值系統

　　內皮素-1是具有21個胺基酸的胜，為目前發現最強的血管收縮物質。血管張力的調控上，內皮素-1扮演了相當重要的角色，並且與其他血管舒張物質及血管收縮物質具有交互作用。其中最主要的是內皮素-1與一氧化氮兩者之間的恆定，並且與腎素-血管張力素系統之間的影響。一氧化氮能導致血管舒張，主要是當血管內皮受到物理性或化學性的刺激時，活化了內皮細胞中的一氧化氮合成，導致下游的訊息傳遞而產生。而血管張力素II則是與內皮素-1最具關聯性的血管收縮物質。過去文獻指出高濃度的內皮素-1會影響內皮細胞正常生理功能的運作，而影響到一氧化氮的產生。另一方面，注射血管收縮素II所引發高血壓的老鼠中，內皮素A型接受器阻斷劑能夠降低腎臟及心血管的損傷。因此我的研究假說，主要是探討高濃度的內皮素，對於血管舒張物質促使一氧化氮釋放，以及腎素-血管張力素系統的表現的影響，藉此來探討高濃度內皮素-1對於心血管系統功能缺失的影響中所可能扮演的角色。利用牛的主動脈內皮細胞及大鼠的血管平滑肌細胞來進行實驗。使用的實驗方法，主要是一氧化氮測定法及西方墨點法。首先，使用內皮素與緩激共同處理內皮細胞，發現緩激所刺激的一氧化氮的產生，大幅的被內皮素所抑制。利用西方墨點法檢測內皮細胞其中蛋白質的表現，發現內皮素是透過抑制一氧化氮合成的磷酸化，來達到抑制一氧化氮產生的效果。並且，高濃度的內皮素-1處理下，使內皮細胞中血管張力素的第一型接受器蛋白表現量上升，且減少血管平滑肌細胞中血管張力素第二型接受器蛋白表現量。本篇論文結果證明，內皮細胞的功能於病理狀態的內皮素-1作用下，扮演較重要的角色，因而內皮功能缺損，極有可能是造成心血管疾病的發生。這些實驗結果發現，有助於更了解內皮素-1在心血管疾病中所扮演的生理角色。

Endothelin-1 (ET-1), a 21-amino acid peptide, is currently known as the most potent vasoconstrictor. ET-1 plays an important role in the regulation of vascular tone and also interacts with other vasoconstrictors and vasodilators. The blood pressure homeostasis is mainly regulated by ET-1 and NO, effecting the rennin-angiotensin system. When vascular endothelial cells receive physical or chemical stimuli, the vascular dilator gas NO is produced by endothelial nitric oxide synthase (eNOS) activation through calcium-dependent or kinase-dependent signaling pathways. The endothelial cell-derived NO induce soluble guanylate cyclase (sGC) activation producing GTP, then decrease in Ca2+ concentration further causes smooth muscle cell (SMC) relaxation. Previous studies indicated that elevated ET-1 level may impair NO production and endothelin type A receptor blocker can prevent cardiovascular and renal effects of angiotensin II. Thus, this study was conducted to investigate the interaction between ET-1, angiotensin and bradykinin upon the NO production in vascular cells and it's mechanism. In this study, we used bovine aortic endothelial cells (BAECs) and smooth muscle cells (SMCs). Greiss reaction was used to detecte NO production by BAECs, and western blot analysis was used to detect the expression level of proteins. Our data showed that ET-1 significantly decreased bradykinin-induced NO production in BAECs. We found that ET-1 impaired the level of bradykinin-induced phosphorylation of eNOS, but ET-1 did not affect the expression of sGC protein. Besides, ET-1 can increase the amount of AT1 receptors in BAECs, and decrease the amount of AT2 receptors in SMCs. In conclusion, this results support that ET-1 impair bradykinin-stimulated eNOS phosphorylation and NO production, and affect expression of angiotensin receptors. These findings will help to elucidate the physiological role of ET-1 in cardiovascular diseases.

中文摘要………………………………………………………………………3
英文摘要………………………………………………………………………5

第一章、文獻回顧…………………………………………………………… 7
一、緒論……………………………………………………………………… 8
二、內皮素…………………………………………………………………… 9
(1)內皮素簡介…………………………………………………………………9
(2)內皮素的生理功能……………………………………………………… 11
(3)內皮素-1與血管反應性…………………………………………………12
三、腎素-血管張力素系統……………………………………………………13
四、緩激……………………………………………………………………15
(1)緩激簡介………………………………………………………………15
(2)緩激的生理功能………………………………………………………15
五、內皮素與一氧化氮之間的關係…………………………………………17
六、內皮素-1與腎素-血管張力素系統之間的關係………………………18

第二章、材料方法……………………………………………………………19
一、研究動機與目的…………………………………………………………20
二、實驗設計…………………………………………………………………21
(1)探討內皮素-1對於緩激刺激一氧化氮分泌量的影響………………21
(2)探討內皮素-1對緩激活化內皮性一氧化氮合成訊息傳遞路徑的影響…………………………………………………………………………22
(3)內皮素-1對血管平滑肌細胞中血管舒張相關蛋白的影響……………22
(4)探討內皮素-1對於內皮細胞及血管平滑肌細胞中血管張力素接受器蛋白表現量的影響…………………………………………………………23
三、牛主動脈內皮細胞與大鼠血管平滑肌細胞……………………………24
四、特定處理藥物與抗體之來源……………………………………………25
五、一氧化氮測定法…………………………………………………………25
六、蛋白質純化………………………………………………………………26
七、蛋白質濃度測定…………………………………………………………26
八、蛋白質電泳………………………………………………………………27
九、西方墨漬法………………………………………………………………28
十、統計分析…………………………………………………………………29

第三章、結果…………………………………………………………………30
一、 單獨或合併處理內皮素-1及緩激對一氧化氮分泌量的影響……31
二、 內皮素-1對於緩激活化內皮性一氧化氮合成訊息傳遞路徑的影響………………………………………………………………………32
三、 內皮素-1對血管平滑肌細胞之SGCα1蛋白表現的影響……………33
四、 內皮素-1對內皮細胞及血管平滑肌細胞之血管張力素接受器蛋白的影響……………………………………………………………………33

第四章、討論…………………………………………………………………35
ㄧ、本研究的主要發現………………………………………………………36
二、內皮素-1對於內皮細胞的影響…………………………………………37
三、內皮素-1對於血管平滑肌的影響………………………………………39
四、結論………………………………………………………………………40

參考文獻…………………………………………………………………… 41
圖…………………………………………………………………………… 53

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