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研究生:陳建志
研究生(外文):Jian Zhi Chen
論文名稱:酒精及內毒素造成心肌細胞氧化壓力與凋亡
論文名稱(外文):Acute Exposure Alcohol and LPS caused Oxidative Stress and Apoptosis in Cardiac myocyte
指導教授:林淑美林淑美引用關係
指導教授(外文):Shu Mei Lin1
學位類別:碩士
校院名稱:中華醫事學院
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:93
語文別:中文
論文頁數:74
中文關鍵詞:心律不整心肌症充血性心臟衰竭氧化壓力細胞凋亡
外文關鍵詞:arrhythmiaheart muscle diseasecongestive heart failureoxidative stressapoptosis
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長期過度攝取酒精會造成心臟之毒性,乃由於酒精所導致心肌功能異常與酒精所引起的心肌疾病,其包含心律不整、心肌症、充血性心臟衰竭。然而西方國家因假日長期濫用酒精,進而導致非缺血性心肌症的發生,嚴重者更進一步造成假日突發性死亡。雖然酒精肝臟毒性機制已確切,但酒精所引起心肌病變之機制仍未清楚。從過去研究得知,氧化壓力在酒精所導致的肝臟損傷中是扮演重要角色之一。臨床上也發現長期酒精濫用會增加血液中內毒素之濃度,並進一步加重肝臟的損傷。因此本實驗主要探討酒精所產生的氧化壓力與內毒素LPS之感染下,是否是造成心肌病變的主要因素。除此之外前人研究指出,細胞凋亡證實在多種不同心臟疾病的病理上扮演重要角色。H9c2 cell是心臟細胞株,是使用於本實驗細胞模式中。細胞以25-50 mM酒精濃度與LPS處理後,培養24、48小時再進行細胞毒性、抗氧化酵素、細胞凋亡分析。細胞毒性實驗(MTT assay)結果顯示,酒精會導致細胞MTT還原能力下降,並造成細胞凋亡的發生。酒精心臟毒性包含降低抗氧化酵素活性SOD,與降低細胞抗氧化物GSH濃度。然而因為抗氧化酵素活性下降,因此進一步以TBARS分析脂質過氧化情形。實驗結果顯示,酒精造成細胞抗氧化酵素活性下降,進而導致脂質過氧化的生成。由本實驗結果得知,酒精心臟毒性可能是因氧化壓力(oxidative stress)與 (apoptosis)所導致。
Cardiac toxicity of excessive alcohol intake has long been recognized. Alcohol can cause abnormal function of heart muscle, known as alcohol cardiomyopathy, and result in arrhythmia, cardiomegaly, congestive heart failure, which in some cases can be life-threatening. For decades, alcohol abuse is a major cause of non-ischemic cardiomyopathy, and is the main factor responsible for post-holiday sudden dead in Western countries. Although the mechanism of alcohol hepatotoxicity has been well elucidated, the molecular mechanism by which alcohol induced cardiomyopathy is still unclear. It has been demonstrated that oxidative stress plays a major part in alcohol-induced liver injury. In clinical cases, chronic alcohol abuse is also associated with the increase of plasma endotoxin level, which may aggravate the oxidative damage in the liver tissue. However, the involvement of oxidative stress, and the effect of endotoxin infection on cadiomyopathy induced by alcohol need further investigation. Furthermore, whether apoptosis is responsible for the loss of myocytes in alcohol-induced cardiac injury waits for elucidation. Therefore, the present study is conducted of clarify the tissue. H9c2 cell, a heart origin cell line, was used as an experiment model in this study. The cell were treated with medium containing different concentration of alcohol (25-50mM), and with or without lipopolysaccharide (LPS). The cell were treated for 24hours or 48hours, followed by determination of the capacity of antioxidant defense system. The result show that alcohol causes H9c2 cell damage assessed by decrease in MTT-reducing activity, and induced cell apoptosis. The alcohol toxicity is associated with the decrease in the activity of antioxidant enzymes, including, superoxide dismutase (SOD), with the reduction of cellular glutathione level. The loss of antioxidant capacity is accompanied by the elevation in lipid peroxidation detected by Thiobarbituric acid reactive substances (TBARS). The data implies the involvement oxidative stress and apoptosis in alcohol cardiac toxicity.
目錄i
圖次iii
誌謝v
縮寫對照表 vi中文摘要vii
Abstract viii第一章文獻回顧 
一、 前言2
二、 酒精代謝途徑與毒性2
三、 氧自由基與抗氧化酵素3四、 酒精與氧化壓力、內毒素的相關性及其作用機轉 5
五、 酒精與細胞凋亡之關係7
六、 酒精與心臟組織相關研究8
七、 實驗目的 11
第二章材料與方法12
一、實驗材料
1、 藥品12
2、 細胞株來源及培養條件13
3、 MTT 呈色法 134、 LDH 活性測定 14
5、 蛋白質濃度測定14
6、 Catalase活性測定 15
7、 GPx活性測定 15
8、 SOD活性測定 16
9、 GSH含量測定 17
10、 TBARS含量測定 17
11、 TUNEL分析 18 12、 統計分析 18
第三章結果 一、酒精之心肌細胞毒性 19
二、酒精對細胞抗氧化系統之影響 21
1、SOD活性之影響 21
2、GPx活性之影響22
3、Catalase活性之影響22
4、GSH濃度之影響22
三、酒精對脂質過氧化之影響  23
第四章討論
1. 酒精的心肌細胞毒性 25
2. 酒精造成心肌細胞凋亡 26
3. 酒精對心肌細胞抗氧化酵素之影響  27
4. 酒精對脂質過氧化之影響 29
參考文獻51
圖次
圖一、酒精與LPS作用24小時對心肌細胞存活率之影響 32
圖二、酒精與LPS作用48小時對心肌細胞存活率之影響 33
圖三、酒精與LPS作用24小時對心肌細胞存活率之影響 34
圖四、酒精與LPS作用48小時對心肌細胞之影響 35
圖五、酒精與LPS造成心肌細胞凋亡 36
圖六、酒精與LPS造成心肌細胞凋亡  37
圖七、酒精與LPS造成心肌細胞凋亡 38
圖八、酒精與LPS造成心肌細胞凋亡 39
圖九、酒精與LPS造成心肌細胞凋亡 40
圖十、酒精與LPS作用24小時對H9c2 cell內SOD活性之
影響 41
圖十一、酒精與LPS作用48小時對H9c2 cell內SOD活性之
影響 42
圖十二、酒精與LPS作用24小時對心肌細胞內GPx活性
影響 43
圖十三、酒精與LPS作用48小時對心肌細胞內GPx活性之
影響 44
圖十四、酒精與LPS作用24小時對心肌細胞內catalase活性之
影響 45
圖十五、酒精與LPS作用48小時對心肌細胞內catalase活性之
影響 46
圖十六、酒精與LPS作用24小時對心肌細胞內GSH含量之
影響 47
圖十七、酒精與LPS作用48小時對心肌細胞內GSH含量之
影響 48
圖十八、酒精與LPS作用24小時對心肌細胞TBARS含量之
影響 49
圖十九、酒精與LPS作用48小時對心肌細胞TBARS含量之
影響 50
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