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研究生:李佩育
研究生(外文):Pei-Yu Lee
論文名稱:在蜘蛛膜下腔出血模式下抑制內皮激素-1對Rho激酶與可溶性鳥酸環化蛋白路徑的影響
論文名稱(外文):Attenuation of Endothelin-1 Regulates Rho-kinase And Soluble Guanylyl Cyclase Pathways After Subarachnoid Hemorrhage
指導教授:關皚麗關皚麗引用關係
指導教授(外文):Aij-Lie Kwan
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:藥理學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:104
中文關鍵詞:蜘蛛膜下腔出血內皮激素-1Rho激酶可溶性鳥酸環化蛋白腦血管痙攣
外文關鍵詞:Subarachnoid HemorrhageEndothelin-1Rho-kinaseSoluble Guanylyl CyclaseCerebral Vasospasm
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蜘蛛膜下腔出血型中風為死亡率極高的出血性腦中風,並且會併發許多嚴重的併發症,例如腦血管痙攣、神經受損與心肺功能異常。許多研究均顯示,內皮激素-1 (endothelin-1;ET-1) 是蜘蛛膜下腔出血後造成腦血管痙攣的主要因子之一。之前實驗的結果顯示,利用內皮激素轉化酶抑制劑─CGS 26303阻斷蜘蛛膜下腔出血後ET-1生成增加的現象,能有效的預防並改善腦血管痙攣的現象。然而,詳細的血管舒張機制尚未明確。
目前已知血管張力的調控機制有:鳥糞嘌呤核苷酸環化酶 (sGC)/環-磷酸鳥嘌呤核苷 (cGMP)/蛋白激酶G (PKG)、RhoA/Rho激酶 (ROCK) 與蛋白激酶C (PKC) 路徑。許多研究已顯示,蜘蛛膜下腔出血後所增加的ET-1會藉由活化PKC路徑,造成腦血管痙攣;然而RhoA/ROCK與sGC/cGMP/PKG路徑在蜘蛛膜下腔出血的動物實驗中較少被討論。本篇研究目的在探討利用CGS 26303抑制蜘蛛膜下腔出血後ET-1的生成,是否是藉由影響RhoA/ROCK與sGC/cGMP/PKG蛋白表現而達緩解腦血管痙攣。
將Sprague-Dawley (SD) 大鼠分為五組:(1) 對照組 (normal control),(2) 蜘蛛膜下腔出血組 (SAH),(3) 蜘蛛膜下腔出血 + 溶媒 (SAH + vehicle),(4) 蜘蛛膜下腔出血 + 預防組 (prevention) 與回復組 (reverse)。其中蜘蛛膜下腔出血,是將SD大鼠的自體血打入腦池中來誘發;預防組與回復組分別誘導蜘蛛膜下腔出血後的1小時與24小時,靜脈注射給予CGS 26303 (1 mg/ml/kg, i.v.)。
SAH模式下利用內皮激素轉化酶抑制劑—CGS 26303,抑制蜘蛛膜下腔出血後ET-1增加,可以改善蜘蛛膜下腔出血所造成的腦血管痙攣現象。蜘蛛膜下腔出血後,基底動脈的RhoA與ROCK表現會增加,sGC、GMP與PKG的表現則降低。投予CGS 26303可以顯著的使RhoA/ROCK與sGC/cGMP/PKG蛋白回復到接近對照組的表現。
本實驗結果顯示,蜘蛛膜下腔出血後增加的ET-1會藉由活化RhoA/ROCK-II與抑制sGC/cGMP/PKG路徑,而造成的血管痙攣。且CGS 26303可經由抑制ET-1的生合成,來改變RhoA/ROCK-II與sGC/cGMP/PKG路徑的表現,達到緩解腦血管痙攣,並改善神經功能受損的作用。此外CGS 26303對腦血管的舒張作用,並不會造成顯著血壓與心跳變化的副作用,亦不會影響多數臟器ROCK-II與sGC的表現。由本實驗可知,內皮激素轉化酶抑制劑—CGS 26303,是極具潛力的血管舒張劑,未來可望運用於蜘蛛膜下腔出血的病人。
Subarachnoid hemorrhage (SAH) is an important subcategory of stroke, because of its unacceptably high levels of mortality, as well as severe complications, such as cerebral vasospasm, neurological deficit and cardiopulmonary abnormality. Many evidences have shown that endothelin-1 (ET-1), a potent vasoconstrictor, plays a pivotal role in cerebral vasospasm following SAH. Previous researches have shown that blocking ET-1 biosynthesis with CGS 26303, an endothelin-converting enzyme (ECE) inhibitor, could effectively prevent and reverse arterial narrowing in SAH animal model; however, the vasorelaxation mechanism is still uncertain.
Vascular smooth muscle tone is regulated by NO-dependent soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG), RhoA/Rho-kinase (ROCK) and protein kinase C (PKC) pathways. Many researches have shown that ET-1 could activate PKC pathway in SAH animal model, while RhoA/ROCK and sGC/cGMP/PKG pathway is discussed rarely. The aim of the study is to exam wherether attenuating ET-1 synthesis in SAH with CGS 26303, could alleviate vasoconstriction through altering RhoA/ROCK and sGC/cGMP/PKG protein expression.
Sprague-Dawley (SD) rats were divided into five groups:(1) normal control, (2) SAH, (3) SAH + vehicle, (4) SAH + prevention and (5) SAH + reverse. CGS 26303 (1 mg/ml/kg, i.v.) was injected into rats at 1 h and 24 h after SAH for the preventive and reverse protocols, respectively. SAH was simulated by single injection of autologous blood into cisterna magna. CGS 26303 could significantly reverse cerebral vasospasm and ET-1 elevation after SAH. The expression of RhoA/ROCK elevated and sGC/cGMP/PKG attenuated in SAH basilar artery. However, CGS 26303 significantly reversed both RhoA/ROCK-II and sGC/cGMP/PKG proteins to control level.
In conclusion, RhoA/ROCK-II and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. Inhibit ET-1 biosynthesis with CGS 26303, could alleviate cerebral vasospasm and neurological deficit after SAH through downregulating of RhoA/ROCK-II expression and upregulating of sGC/cGMP/PKG expression. Besides, unwanted blood pressure and heart rate changes did not occur after vasorelaxation with CGS 26303. Moreover, ROCK-II and sGC expression were not affected either.
With this study, we found that CGS 26303 is a potential drug for prevention and reverse vasospasm after SAH. Adverse effects were rarely found in our experiments, therefore, CGS 26303 could be expected to use in SAH patients in the future.
中文摘要.......................................... 1
英文摘要.......................................... 3
縮寫表............................................ 6
第一章 序言--------------------------------------- 8
第一節 腦中風的分類與流行病學.................... 8
第二節 蜘蛛膜下腔出血............................ 9
第三節 內皮激素..................................11
第四節 腦血管分佈與血液供給......................15
第五節 蜘蛛膜下腔出血所造成腦血管痙攣的相關機制..16
第六節 一氧化氮與內皮激素的對腦血管張力的調控....20
第七節 蜘蛛膜下腔出血後的併發症..................22
第八節 實驗藥物―CGS 26303........................23
第二章 研究材料-----------------------------------26
第三章 研究方法-----------------------------------38
第四章 實驗結果-----------------------------------49
第五章 討論---------------------------------------58
第六章 附圖與附表---------------------------------66
第七章 參考文獻-----------------------------------88
第七章參考文獻

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