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研究生:郭倩妤
研究生(外文):Qian-YuKuok
論文名稱:靈芝酸對於乙型交感神經受體素造成心肌受損之保護作用
論文名稱(外文):The protective effect of Ganoderic acids against myocardial injury induced by β-adrenoceptor agonists
指導教授:莫凡毅
指導教授(外文):Fan-E Mo
學位類別:碩士
校院名稱:國立成功大學
系所名稱:細胞生物及解剖學研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:50
中文關鍵詞:乙型交感神經受體素心臟受損心肌梗塞靈芝酸細胞凋亡
外文關鍵詞:β-adrenoceptor agonistsCardiac injurymyocardial infarctionGanoderic acidsApoptosis
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  • 下載下載:18
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乙型交感神經受體素,可促使平滑肌鬆弛、增強心臟肌肉的收縮和速率,因此臨床上這類藥物經常被使用於緩解支氣管痙攣治療氣喘或慢性阻塞性肺病這類疾病。
β1-腎上腺素受體素 (ISO)為乙型交感神經受體素的一種,其臨床主要是透過用於治療心速過緩以及心臟傳導阻滯疾病。有研究指出,當小鼠長期受到ISO刺激,會誘發心肌細胞凋亡,造成心肌損傷,本篇研究旨在探討經由乙型交感神經受體素誘導心臟受損的治療策略。
不論是古代或是現代的藥學及臨床研究,皆顯示靈芝具有抗氧化以及長生的功效。過去有研究指出,靈芝可保護心臟免於受到ISO所造成的心肌損傷,但究竟是靈芝的何種成分對於心臟具保護效果目前是不清楚的。我們推測靈芝的萃取物-靈芝酸可能是主要保護心臟免於受到ISO刺激所造成傷害之重要成分,同時將探討其分子機轉。為了驗證此假設,建立C57BL6小鼠模型皮下ISO100 mg/kg/day,每天一次,連續五天,同時在給ISO前2小時口服餵食靈芝粉300mg/kg/day,等待兩天後犧牲。另一急性藥物處理BALB/c小鼠模型,同時處理ISO 10mg/kg (皮下注射一劑)以及靈芝酸500mg/kg/day (每日一劑,連續處理2天),等待48小時後犧牲。將心臟組織利用蘇木紫與署紅、Masson氏三色染色法進行心臟病理分析;接著利用TUNEL染色法檢測心肌細胞凋亡的情形,而後更進一步利用免疫組織化學染色法觀察促死亡因子FasL之表現量;並且在動物犧牲前利用心電圖檢測其心臟功能。
結果顯示,在ISO的刺激下會導致明顯的心臟受損及產生纖維化,並且促使心肌細胞透過促死因子FasL走向凋亡。值得注意的是,上述這些心肌梗塞、纖維化以及心肌細胞凋亡,在靈芝/靈芝酸的處理之下皆受到了保護。心電圖的檢測當中發現,在ISO的處理之下R峯以及ST段明顯的升高且延長,表示心臟有缺血性壞死的情形產生,這樣的結果在靈芝的處理之下是看不到的。這些結果說明,靈芝及靈芝酸可有效防止ISO所誘導的心肌梗塞。
本篇研究希望能建立將靈芝純化物-靈芝酸,作為一種新型天然保健藥物,用以保護乙型交感神經受體素類藥物所誘發的心肌損傷。

β-adrenoceptor agonists promote relaxation in smooth muscle and increase contractility and pulse rates in cardiac muscle. Therefore, they are often used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary disease.
Isoproterenol (ISO) is one of the sympathomimetic beta adrenoceptor agonists. Its primary use is for bradycardia, heart block. Long-term stimulation by ISO may induce cardiomyocyte apoptosis, leading to myocardial injury in mice. We intended to explore therapeutic strategies for heart injury-induced by ISO. Ancient and modern pharmacological and clinical studies have shown that Ganoderma tsugae (GT) possesses effects of antioxidant and for longevity. Previous research has shown that GT can prevent myocardial injury induced by ISO. But the active components of GT to the heart is still unknown. We examined whether purified Ganoderic acids (GAs) from GT maybe responsible for protecting hearts from ISO-induced cardiac injury and its molecular mechanism.
To test this hypothesis, GT was administered to mice in an ISO-induced myocardial injury model. Adult C57BL6 mice were injected with ISO (100 mg/kg, s.c.) once daily for 5 days, and the mice received oral administration GT 300mg/kg/day for 1week . Hearts were collected from mice 2 days after the course of ISO treatments. In a more acute model, adult BALB/c mice were injected with single dose of ISO (10 mg/kg, s.c.), in combination with the administration of crude extract of GAs (500mg/kg/day, s.c.) once daily for 2 days. Hearts were collected from mice 2 days after the injection. H&E and trichrome staining were performed to analyze the pathology of the hearts. Other procedures including, cardiac cell TUNEL staining for apoptosis, immunohistochemistry staining for pro-apoptosis factor, FasL,and electrocardiography (EKG) for cardiac physiology, were carried out as well.
ISO treatments resulted in obvious myocyte loss, cardiac damage and fibrosis. In addition, ISO caused cardiac cell apoptosis through FasL. Remarkably, the myocardial infarction (MI), fibrosis and cell death-induced by ISO were not detected in mice receiving GT or GAs. We further used EKG to access the heart function in mice. The result showed that ISO enlarged R peaks significantly and ST intervals were obviously increased and prolonged. Combination of ISO and GTsustained normal heart function in mice. These results demonstrated that GT and GAs effectively prevented from ISO-induced MI.
We hope to establish GT and GAs as novel therapeutic strategies for myocardial injury induced by β-adrenoceptor agonists.

摘要------------------------------------------------i
Abstract-------------------------------------------iii
致謝------------------------------------------------v
目錄------------------------------------------------vii
圖表目錄---------------------------------------------x
縮寫表-----------------------------------------------xi
第一章 前言------------------------------------------1
1-1乙型交感神經受體素(β-adrenoceptor agonists)---------1
1-2靈芝(Ganoderma tsugae)---------------------------3
1-3 細胞凋亡 Apoptosis-------------------------------5
研究動機---------------------------------------------7
第二章 材料與方法--------------------------------------8
2-1 材料--------------------------------------------8
2-1-1 藥品配製/化學藥品--------------------------------8
2-1-2 實驗材料---------------------------------------12
2-1-3 儀器設備---------------------------------------12
2-2方法---------------------------------------------13
2-2-1動物-------------------------------------------13
2-2-2 組織與切片的製備--------------------------------16
2-2-3 組織染色---------------------------------------17
第三章 結果------------------------------------------24
3-1 C57BL6小鼠動物模式--------------------------------24
3-1-1 靈芝保護心臟免於β1-腎上腺素受體激活劑 isoproterenol(ISO)造成的心肌梗塞-------------------------------------------24
3-1-2靈芝保護心臟免於ISO造成的心肌細胞凋亡----------------24
3-1-3 靈芝保護保護心臟免於ISO造成的心臟功能受損------------25
3-2 BALB/c小鼠動物模式--------------------------------26
3-2-1 靈芝酸保護心臟免於ISO造成的心肌梗塞-----------------26
3-2-2 靈芝酸保護心臟免於ISO造成的心肌細胞凋亡--------------26
3-2-3 靈芝酸保護心臟免於受到ISO透過促死亡因子FasL所造成之心臟損傷--------------------------------------------------------27
3-2-4 靈芝酸保護心臟免於受到ISO透過促死亡因子Fas 所造成之心臟損傷--------------------------------------------------------28
3-2-5 靈芝酸降低ISO所誘發之促死亡因子Fas-----------------28
3-3-1 萊克多巴胺誘發心肌損傷----------------------------29
第四章 討論-------------------------------------------30
第五章 結論-------------------------------------------34
第六章 參考文獻----------------------------------------35
第七章 圖表-------------------------------------------40
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