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研究生:蔡丹卿
研究生(外文):Dan-Chin Tsai
論文名稱:綠茶萃取物於活體動物左冠狀動脈結紮引發心肌梗塞的保護作用
論文名稱(外文):A STUDY IN VIVO WITH THE CARDIOPROTECTION OF GREEN TEA EXTRACTS ON MYOCARDIAL INFARCTION INDUCED BY CORONARY LIGATION OF RAT HEART
指導教授:劉英明
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:60
中文關鍵詞:綠茶多酚兒茶素心肌梗塞抗纖維化保護心臟細胞 凋亡STAT-1活化酸化鈣離子敏感性肌原纖維水解ATP酵素活性
外文關鍵詞:green tea polyphenolcatechinmyocardialinfarctionantifibroticcardioprotective agentsischemia/reperfusion animal modelapoptosisSTAT-1 activationacidosiscalcium sensitivitymyofibrillar actomyosin ATPase activity
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茶一年消耗近30億公斤。近期綠茶更是廣被使用於心血管及癌症的相關研究。日本學者經過長期研究發現:飲用綠茶者可有效降低死亡率及因心血管死亡的機率。綠茶素中含有多酚類的化學結構,具有抗氧化能力,以及清除細胞內的自由基。利用缺血/灌流的動物離體心臟研究,顯示心臟缺血會誘發心臟細胞內轉錄因子STAT-1的活化路徑,造成心肌細胞之凋亡;而相同模式處理綠茶多酚能有效抑制心肌內STAT-1的活化路徑。另外,將大鼠進行腹主動脈狹窄手術產生壓力以及H2O2誘發心肌細胞產生凋亡的研究報告,發現經過綠茶素處理過後的心肌細胞能有效抑制p53增加及bcl-2減少,並且降低氧化所產生的細胞凋亡。然而,目前並沒有活體實驗動物證據,可直接證明綠茶素如何在心肌細胞內保護細胞免於傷害。因此,我們利用大鼠(300-350克)進行冠狀動脈左前降支結紮手術,模擬急性心肌梗塞模式,探討綠茶對於心臟缺血時的保護作用。首先動物手術後,可經由肉眼觀察心臟缺血象徵。另外手術後,抽血確認血液cTn I產生裂解測量以及測試生理值變化及心臟超音波。實驗結果顯示:動物手術後,血液中cTn I在2-28小時有明顯增加,實驗動物在手術後三天有血壓下降、心跳加快的變化;心臟超音波掃瞄也顯示實驗動物之心臟收縮功能受到影響。之後,實驗動物開始餵食有效劑量的綠茶萃取物(400mg/kg),分別在3天、1、3、7周測量心臟超音波;以及3和7週後取心臟進行組織切片觀察,以探討綠茶多酚對於心肌梗塞之實驗動物心臟的保護作用的評估。組織切片,分別以H&E染色、Masson`s trichrome染色及OPN組織免疫染色分析心臟纖維化和TTC染色分析梗塞區,結果顯示餵食過綠茶萃取物的老鼠心臟和沒有餵藥的老鼠心臟相比有回復的效果。有餵藥的老鼠和沒餵藥老鼠比較,產生纖維化減少33%,有餵藥的老鼠和沒餵藥老鼠比較梗塞區回復65%;依據無餵綠茶萃取物組在梗塞後左心室腔室大小、收縮及肌肉壁層厚度相較,綠茶萃取物組有顯著改善效用。綜觀以上結果證實綠茶多酚對於心肌梗塞後的心臟具有保護的功效。
A recent study showed that green tea consumption is associated with reduced mortality resulting in all causes and cardiovascular disease. Green tea and its constituent catechins are best known for their antioxidant properties for removing free radical. Previously, it was demonstrated that STAT-1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following the ischemia/reperfusion (I/R) injury. EGCG treatment reduced STAT-1 phosphorylation and hence protected cardiac myocytes against I/R-induced apoptotic cell death. In addition, EGCG was shown to prevent cardiomyocyte from oxidative stress induced cell apoptosis in vitro. The mechanism is postulated to be related to the inhibitory effects of EGCG on p53 induction and bcl-2 decreased expression. Thus far there is no evidence for green tea in vivo. With this aim, we used the rat (300-350g) as an animal model for the surgery of ligation of left anterior descending artery (LAD) to simulate a pathological condition of myocardial infarction (MI). Firstly, we confirmed that LAD igation induced myocardial infarction by measuring the blood levels of cardiac Troponin I (cTnI) as a molecular marker for MI. The operated rats followed by LAD ligation for 3 day, had decreased blood pressure and increased heart rate and had affected contraction function by echocardiograms. This result suggested that myocardial damage disturbed the normal function of cardiovascular system in the LAD-ligated animals. In addition, histological observations by TTC ischemia staining, H & E staining, Masson’n Trichrome stain and histo- immuno-chemistry against osteopontin (OPN) staining suggested that remodeling of myocardium as seen by fibrosis and expression of osteopontin (OPN) occurred in the hearts of animals after 2-, 3- and 7- weeks ligation. In addition, echocardiograms showed that impairment of myocardial functions occurred in post-MI rats, but the cardiac functions were recovered after 3 days,1-, 3- and 7- weeks ligation. Three to seven weeks had beneficial effect significantly decreases 65% in infarction size, decreases in the expression of OPN, and decreases 32% in the degree of fibrosis in damaged cardiac tissues. Deterioration in LV chamber size, systolic function and wall thickness of infarction region were found in the group without GTE treatment after LAD ligation. The recovering effect was significant after the GTE treatment. Taken together, the data reported here provides evidence in support for the beneficial effects of green tea polyphenols on protection of myocardial damages in MI animals.
壹、前言
一、研究概要............................................1
二、 綠茶萃取物成分與機能...............................2
三、流行病學研究........................................3
四、心肌梗塞............................................4
五、心臟肌肉蛋白及收縮分子開關..........................7
六、.酸化(Acidosis)對於心臟的影響.......................9
(一)心臟肌肉細胞機械功能方面........................9
(二)心肌細胞凋亡(apoptosis).......................10
七、Osteopontint產生...................................13
八、綠茶萃取物對於受損心臟的影響.......................14
九、研究方向...........................................15
貳、材料與方法
一、實驗流程...........................................16
二、實驗動物...........................................18
三、綠茶萃取物.........................................18
四、建立心肌梗塞之動物模式............................18
五、血液樣品採集......................................19
六、西方轉漬法(Western blot)..........................19
七、術後生理值之測量..................................21
八、心臟超音波........................................21
九、心臟組織取樣......................................23
十、組織包埋和切片....................................23
十一、H&E染色法.......................................23
十二、Masson’s Trichrome 染色........................23
十三、OPN組織免疫染色.................................24
十四、TTC染色.........................................24
十五、統計分析........................................25
參、實驗結果
一、確認心肌梗塞模式建立之徵象.........................26
(一) LAD結紮後心臟缺血徵象.......................26
(二) 手術後血液生化檢查..............................26
(三)LAD結紮手術對於心臟功能及生理值的影響...........27
(四)LAD結紮手術對於心臟組織方面的影響...............29
二、餵食綠茶萃取物對於心肌梗塞之梗塞區大小的影響.......30
三、組織切片染色分析綠茶萃取物對心肌梗塞之保護作用
(一)手術三周後組織病理變化..........................33
(二)手術七周後組織病理變化..........................36
四、心臟超音波分析綠茶萃取物對於心肌梗塞的影響.........38
(一)左心室舒張末期前壁厚度(IVSd)...................38
(二)左心室舒張末期內徑(LVIDd).....................39
(三)左心室內徑周長縮短分率(FS)....................40
(四)左心室射出分率(EF)............................41
肆、討論
一、LAD結紮手術對心臟的影響 ............................45
二、綠茶萃取物對於受損心臟的作用.......................46
(一)組織學方面)....................................47
(二)心臟功能方面...................................48
三、為何手術3天後餵綠茶萃取物仍有效...................51
四、結論 ..............................................52
五、未來展望......................................52
參考文獻...............................................53
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