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研究生:黃啟彰
研究生(外文):Chi-Chang Huang
論文名稱:以Lieber-DeCarli之動物模式探討慢性酒精毒性對於抗氧化狀態及肝臟形態變化之影響
論文名稱(外文):Effects of Chronic Alcoholic Toxicity on Antioxidative Status and Hepatic Morphologic Changes by Lieber-DeCarli Animal Model
指導教授:楊素卿楊素卿引用關係
指導教授(外文):Suh-Ching Yang
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:藥學系
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:223
中文關鍵詞:酒精性別差異四氯化碳脂解酶雙醣酶氧化壓力脂質過氧化P450 2E1黃嘌呤氧化酶骨髓過氧化酶脂肪肝脂肪變性硫巴比妥酸過氧化氫四氯化碳胺酸轉胺羥基丁酸
外文關鍵詞:ethanolgender differencescarbon tetrachloridelipasedisaccharidaseoxidative stresslipid peroxidationCYP2E1xanthin
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本研究利用Lieber-DeCarli流質飲食模式,探討不同性別於酒精性肝臟疾病方面之表現。此外,本研究也針對酒精與四氯化碳所造成之肝傷害進行探討並作系統性之比較。
首先,在第一部份,不同性別對於長期攝取酒精所造成肝損傷之影響的研究中,使用年齡相同之雄性與雌性Wistar品系大白鼠,根據Lieber-DeCarli模式,分別餵食對照或含有酒精的液體飼料,為期12週。將40隻大白鼠依照血漿中肝功能指數天門冬胺酸轉胺酶(aspartate aminotransferase, AST)與丙胺酸轉胺酶(alanine aminotransferase, ALT)之活性分成4組:雄性對照組(MC)、雄性酒精組(ME)、雌性對照組(FC)以及雌性酒精組(FE),每組10隻。餵食酒精12週之後,結果顯示,長期餵食酒精會造成顯著的影響包括:增加相對肝重(%),提高血漿中AST與ALT之活性,增加血漿中總膽固醇(total cholesterol, TC)、高密度脂蛋白膽固醇(high density lipoprotein cholesterol, HDL-C)、乳酸(lactate)、游離脂肪酸(non esterified fatty acid, NEFA)、羥基丁酸(-hydroxy- butyrate)與腫瘤壞死因子(tumor necrosis factor-, TNF-)之濃度,增加肝臟中三酸甘油酯(triglyceride, TG)與TC之含量,誘導肝臟中細胞色素P450 2E1 (CYP2E1)蛋白質之表現,提高肝臟中黃嘌呤氧化酶(xanthine oxidase, XO)與骨髓過氧化酶(myeloperoxidase, MPO)之活性,增加血漿與肝臟中脂質過氧化產物即硫巴比妥酸反應物質(thiobarbituric acid reactive substances, TBARS)之含量,減少肝臟中還原型麩胱甘肽(glutathione, GSH)之含量並降低還原型麩胱甘肽/氧化型麩胱甘肽(oxidized glutathione, GSSG)之比值,降低肝臟中抗氧化酵素麩胱甘肽過氧化酶(glutathione peroxidase, GPX)、過氧化氫酶(catalase, CAT)與超氧化物歧化酶(superoxide dismutase, SOD)之活性,提高空腸中脂解酶(lipase)之活性,降低空腸中雙醣酶(sucrase、maltase與lactase)之活性,而且病理學上明顯有脂肪肝形成之情形。此外,相較於雄性組,雌性組大白鼠血漿中AST活性明顯升高,血漿中NEFA與-hydroxybutyrate濃度顯著增加,肝臟中TG與TC含量明顯增加,肝臟中TBARS含量顯著增加,肝臟中GSH含量明顯減少、GSH/GSSG比值顯著下降,肝臟中抗氧化酵素GPX與CAT之活性均顯著降低,而且脂肪肝程度較嚴重。由此可知,不論雄性或雌性,長期攝取酒精都會造成空腸中消化酵素活性明顯受到影響。另外,長期攝取酒精也會導致肝功能指標上升、脂質代謝異常、氧化壓力上升、脂質過氧化產物增加、抗氧化物質含量減少、抗氧化酵素活性降低以及病理上造成肝損傷;其中又以雌性組所造成的影響更嚴重。
在第二部份,比較酒精與四氯化碳造成肝傷害之實驗中,以雄性Wistar大白鼠50隻作為實驗動物,依肝功能指標AST與ALT分成5組:對照組(C)、酒精組(E)、酒精治療組(ES)、四氯化碳組(CCL)以及四氯化碳治療組(CCLS),每組10隻,實驗期為12週。結果顯示,長期餵食酒精或注射四氯化碳都會造成的影響包括:肝重與相對肝重(%)明顯增加,血漿中AST與ALT活性於實驗期第2、4、6、8、10、12週皆明顯上升,血漿中TG濃度於實驗期第10、12週顯著減少,肝臟中TG與TC含量均顯著增加,肝臟中CYP2E1蛋白質表現量明顯受到誘導,肝臟中MPO活性顯著上升,血漿中TBARS濃度於實驗期第2、4、6、8、10、12週皆明顯增加,肝臟中TBARS含量顯著增加,肝臟中GSH含量顯著減少、GSH/GSSG比值明顯下降,肝臟中抗氧化酵素GPX與SOD活性皆顯著降低,而且病理學上明顯有脂肪肝與脂肪變性形成之情形。此外,於實驗結束後,相較於酒精組,四氯化碳組大白鼠肝重與相對肝重(%)均明顯增加,血漿中AST與ALT之活性均明顯上升,血漿中TG、TC與HDL-C濃度顯著減少,肝臟中TG與TC之含量均顯著增加,肝臟中XO與MPO活性均顯著升高,血漿中TBARS濃度明顯增加,肝臟中GSH含量顯著減少、GSSG含量顯著增加、GSH/GSSG比值明顯下降,肝臟中抗氧化酵素麩胱甘肽還原酶(glutathione reductase, GRD)與CAT活性皆顯著降低,而且脂肪肝與脂肪變性之情形更嚴重。由此可知,酒精與四氯化碳都會造成肝功能指標上升、脂質代謝異常、氧化壓力上升、脂質過氧化產物增加、抗氧化物質含量減少、抗氧化酵素活性降低以及病理上造成肝損傷;其中又以四氯化碳所造成的影響更嚴重。
This dissertation had two major parts; one was to evaluate the correlation between gender differences and chronic alcoholic liver disease. In addition, effects of gender differences on jejunal lipase and disaccharidase activities, the liver function tests, metabolic disorders and oxidative damage were also examined. Another major part was to examine oxidative stress of alcoholic injury and carbon tetrachloride- induced damage in relation to risk factors for liver disease.
The first part of this study was to investigate the effects of gender differences on alcoholic liver disease in rats with chronic ethanol consumption. Age-matched male and female Wistar rats were fed control or ethanol-containing liquid diets for 12 weeks following the Lieber-DeCarli model. According to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, 40 rats were divided into four groups as follows: male control group (MC), male ethanol group (ME), female control group (FC), and female ethanol group (FE). After ethanol feeding for 12 weeks, the findings indicated significant main effects of ethanol consumption on increased relative liver weight (%); elevated plasma AST and ALT activities; raised plasma total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), lactate, non-esterfied fatty acid (NEFA), -hydroxybutyrate, and tumor necrosis factor-alpha (TNF-) concentrations; increased hepatic triglyceride (TG) and TC contents; induction of hepatic microsomal cytochrome P450 2E1 (CYP2E1); elevated hepatic xanthine oxidase (XO) and myeloperoxidase (MPO) activities; increased both plasma and hepatic thiobarbituric acid reactive substances (TBARS) levels; reduced hepatic glutathione (GSH) content and the ratio of GSH/oxidized GSH (GSSG); decreased hepatic antioxidant enzymes, glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) activities; elevated jejunal lipase activity; lowered all jejunal disaccharidase, sucrase, maltase and lactase, activities; and the formation of fatty liver; when compared to control group. Furthermore, the results also showed significant main effects of gender differences on elevated plasma AST activity; increased both plasma NEFA and -hydroxybutyrate concentrations; augmented hepatic TG and TC contents; raised hepatic TBARS level; reduced hepatic GSH content and the ratio of GSH/GSSG; decreased hepatic antioxidant enzymes, GPX and CAT activities; and degree of fatty liver; when compared to male group. In conclusion, our results suggest that long-term ethanol consumption significantly increased jejunal lipase and decreased jejunal disaccharidase (sucrase, maltase, and lactase) activities in both male and female rats. Our results also show that chronic ethanol administration induced a greater susceptibility to liver damage in female rats than in male rats.
In the second part, we examined the effects of ethanol- or carbon tetrachloride-induced liver injury in male Wistar rats following the Lieber-DeCarli liquid diet. According to both the plasma AST and ALT activities, 50 rats were assigned to five groups: C (control feeding), E (ethanol feeding), ES (ethanol feeding combined with the supplementation of silymarin, 200 mg/kg BW/day), CCL (CCl4 injection and control feeding) and CCLS (CCl4 injection and control feeding combined with the supplementation of silymarin, 200 mg/kg BW/day). Rats in groups CCL and CCLS were intraperitoneally injected with 0.75 mL/kg BW of 40% CCl4 dissolved in olive oil once a week, while rats in groups C, E and ES were intraperitoneally injected with 0.75 mL/kg BW of olive oil only. Our dada indicated significant main effects of both ethanol feeding and carbon tetrachloride injection on increased relative liver weight (%); elevated plasma AST and ALT activities at weeks 2, 4, 6, 8, 10 and 12; lowered plasma TG concentrations at weeks 10 and 12; increased hepatic TG and TC contents; induction of hepatic microsomal CYP2E1; elevated hepatic MPO activity; increased plasma TBARS concentrations at weeks 2, 4, 6, 8, 10 and 12; increased hepatic TBARS level; reduced hepatic GSH content and the ratio of GSH/GSSG; decreased hepatic antioxidant enzymes, GPX and SOD, activities; and pathologically changed liver; when compared to control feeding. Moreover, after 12 weeks, the results also showed significant main effects of carbon tetrachloride injection on increased relative liver weight (%); elevated plasma AST and ALT activities; reduced all the plasma TG, TC and HDL-C concentrations; augmented hepatic TG and TC contents; elevated hepatic XO and MPO activities; increased plasma TBARS concentration; reduced hepatic GSH content and the ratio of GSH/GSSG, whereas increased GSSG level; decreased hepatic antioxidant enzymes, GRD and CAT activities; and severe fatty change in livers; when compared to ethanol feeding. In conclusion, our results suggest that both long-term ethanol feeding and carbon tetrachloride injection significantly increased oxidative stress, lipid peroxidation, and decreases in GSH concentrations and the ratio of GSH/GSSG in rats. Our results also show that carbon tetrachloride injection induced a greater susceptibility to liver damage than ethanol feeding.

Key words: ethanol, gender differences, carbon tetrachloride, lipase, disaccharidase, oxidative stress, lipid peroxidation, CYP2E1, xanthine oxidase, myeloperoxidase, fatty liver, fatty degeneration
中文摘要………………………………………………………………… I
英文摘要………………………………………………………………… IV

致謝……………………………………………………………………… VII
目錄……………………………………………………………………… VIII

表目次…………………………………………………………………… XI
圖目次…………………………………………………………………… XIV

第一章 緒論………………………………………………………………1

第二章 文獻回顧…………………………………………………………4
第一節 肝臟的生理結構與功能………………………………………4
一、肝臟的構造與生理功能…………………………………………4
二、構成肝臟的細胞種類及其生理作用……………………………6
第二節 酒精性肝臟疾病形成之原因…………………………………9
一、酒精對於脂質代謝之影響………………………………………9
二、酒精對於肝臟氧化傷害之影響…………………………………10
三、酒精代謝產物乙醛之毒性作用…………………………………22
四、細胞激素(cytokine)與酒精性肝臟疾病之關係…………………25
五、內毒素(endotoxin)與酒精性肝臟疾病之關係…………………..26
六、不同性別在酒精性肝臟疾病方面之表現………………………..30
第三節 酒精性肝臟疾病動物模式之相關研究……………………… 32
一、Lieber-DeCarli model……………………………………………32
二、Tsukamoto-French model………………………………………33
第四節 四氯化碳造成肝損傷之作用機轉…………………………… 35
一、CCl4會促進氧化壓力形成而導致肝損傷………………………35
二、CCl4會造成脂質代謝異常而導致肝臟脂肪堆積………………38
三、CCl4會造成肝臟纖維化之形成…………………………………40
第五節 四氯化碳誘導肝損傷動物模式之相關研究…………………41
一、CCl4誘導肝損傷之方式……………………………………….. 41
二、CCl4所誘導之肝損傷於肝臟疾病方面的應用……………….. 42
第六節 保肝藥物Silymarin之相關研究……………………………..43
一、Silymarin之介紹……………………………………………….. 43
二、Silymarin與酒精性肝臟疾病之相關研究………………………44
三、Silymarin與四氯化碳誘導肝損傷之相關研究…………………46

第三章 不同性別對於長期攝取酒精所造成肝損傷之影響……………48
第一節 研究方法………………………………………………………48
一、實驗材料…………………………………………………………48
二、分析項目與測量方法……………………………………………52
三、統計分析方法……………………………………………………74
第二節 結果……………………………………………………………75
一、肝功能指數與肝臟病理觀察之結果……………………………75
二、空腸中消化酵素活性以及十二指腸病理觀察之結果…………77
三、脂質代謝之結果…………………………………………………78
四、其他血液生化值之結果…………………………………………79
五、造成氧化壓力上升相關酵素活性之結果………………………80
六、脂質過氧化之結果………………………………………………82
七、抗氧化物質含量之結果…………………………………………83
八、抗氧化酵素活性之結果…………………………………………86
第三節 討論……………………………………………………………89
一、肝功能指數與肝臟病理觀察方面………………………………89
二、空腸中消化酵素活性以及十二指腸病理觀察方面……………92
三、脂質代謝方面……………………………………………………95
四、其他血液生化值方面……………………………………………99
五、造成氧化壓力上升相關酵素活性方面…………………………103
六、脂質過氧化方面…………………………………………………106
七、抗氧化物質含量方面……………………………………………107
八、抗氧化酵素活性方面……………………………………………109
第四節 結論…………………………………………………………...111

第四章 酒精性肝損傷與四氯化碳肝傷害之系統性比較……………..112
第一節 研究方法……………………………………………………..112
一、實驗材料………………………………………………………..112
二、分析項目與測量方法……………………………………………115
三、統計分析方法……………………………………………………116
第二節 結果…………………………………………………………..118
一、肝功能指數與肝臟病理觀察之結果……………………………118
二、脂質代謝之結果…………………………………………………123
三、造成氧化壓力上升相關酵素活性之結果………………………129
四、脂質過氧化之結果………………………………………………131
五、抗氧化物質含量之結果…………………………………………133
六、抗氧化酵素活性之結果…………………………………………136
第三節 討論…………………………………………………………. 139
一、肝功能指數與肝臟病理觀察方面………………………………139
二、脂質代謝方面……………………………………………………141
三、造成氧化壓力上升相關酵素活性方面…………………………144
四、脂質過氧化方面…………………………………………………146
五、抗氧化物質含量方面……………………………………………147
六、抗氧化酵素活性方面……………………………………………148
第四節 結論…………………………………………………………..149

第五章 總結……………………………………………………………..150

第六章 參考文獻………………………………………………………..152
表目次
表一、不同性別大白鼠長期餵食酒精液體飼料對於初始體重、期末體重、肝臟總重以及相對肝臟重量(%)之影響………………………...173
表二、不同性別大白鼠長期餵食酒精液體飼料對於實驗初與實驗末血漿中肝功能指數AST與ALT活性之影響…………………………174
表三、不同性別大白鼠長期餵食酒精液體飼料對於空腸中脂解酶以及雙醣酶活性之影響………………………………………………… 175
表四、不同性別大白鼠長期餵食酒精液體飼料對於實驗末血漿中TG、TC、LDL-C與HDL-C濃度之影響………………………………176
表五、不同性別大白鼠長期餵食酒精液體飼料對於肝臟中TG與TC含量之影響…………………………………………………………… 177
表六、不同性別大白鼠長期餵食酒精液體飼料對於實驗末其他血漿中生化濃度之影響…………………………………………………… 178
表七、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中XO活性之影響…………………………………………………………… 179
表八、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中MPO活性之影響…………………………………………………………… 180
表九、不同性別大白鼠長期餵食酒精液體飼料對於血漿中脂質過氧化產物TBARS濃度之影響………………………………………… 181
表十、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中脂質過氧化產物TBARS含量之影響…………………………………… 182
表十一、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化物質GSH含量之影響………………………………………...183
表十二、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化物質GSSG含量之影響……………………………………… 184
表十三、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化物質GSH/GSSG比值之影響………………………………… 185
表十四、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化酵素GPX活性之影響………………………………………… 186
表十五、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化酵素GRD活性之影響………………………………………… 187
表十六、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化酵素CAT活性之影響………………………………………… 188
表十七、不同性別大白鼠長期餵食酒精液體飼料對於不同組織中抗氧化酵素SOD活性之影響…………………………………………189

表十八、長期餵食酒精或注射四氯化碳對於大白鼠初始體重、期末體重、每日體重增加量、肝臟重量以及相對肝臟重之影響…………190
表十九、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠體重變化之影響……………………………………………………………..191
表二十、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中肝功能指數AST活性之影響………………………………………192
表二十一、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中肝功能指數ALT活性之影響…………………………………193
表二十二、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中TG濃度之影響…………………………………………………194
表二十三、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中TC濃度之影響…………………………………………………195
表二十四、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中低密度脂蛋白膽固醇濃度之影響……………………………196
表二十五、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中HDL-C濃度之影響…………………………………………197
表二十六、長期餵食酒精或注射四氯化碳對於大白鼠肝臟中TG與TC含量之影響……………………………………………………198
表二十七、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中XO活性之影響……………………………………………………199
表二十八、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中MPO活性之影響……………………………………………………200
表二十九、長期餵食酒精或注射四氯化碳對於實驗期間大白鼠血漿中脂質過氧化產物TBARS濃度之影響………………………201
表三十、長期餵食酒精或注射四氯化碳對於大白鼠組織中脂質過氧化產物TBARS含量之影響……………………………………202
表三十一、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中GSH含量之影響……………………………………………………203
表三十二、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中GSSG含量之影響…………………………………………………204
表三十三、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中GSH/GSSG比值之影響……………………………………205
表三十四、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中抗氧化酵素GPX活性之影響………………………………………206
表三十五、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中抗氧化酵素GRD活性之影響………………………………………207
表三十六、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中抗氧化酵素CAT活性之影響………………………………………208
表三十七、長期餵食酒精或注射四氯化碳對於大白鼠不同組織中抗氧化酵素SOD活性之影響………………………………………209
圖一、餵食酒精之後,液體飼料(A)與大白鼠血液(B)中酒精濃度變化之影響…………………………………………………………………210
圖二、不同性別大白鼠長期餵食酒精液體飼料對於肝臟病理切片結果之影響………………………………………………………211
圖三、不同性別大白鼠長期餵食酒精液體飼料對於十二指腸病理切片結果之影響…………………………………………………212
圖四、不同性別大白鼠長期餵食酒精液體飼料對於肝臟中微粒體細胞色素P450 2E1蛋白質表現之影響…………………………213

圖五、長期餵食酒精或注射四氯化碳對於大白鼠肝臟組織外觀之影響…………………………………………………………………214
圖六a、C組大白鼠肝臟病理切片圖(H&E stain) ………………………215
圖六b、E組大白鼠肝臟病理切片圖(H&E stain) ………………………216
圖六c、ES組大白鼠肝臟病理切片圖(H&E stain) ……………………217
圖六d、CCL組大白鼠肝臟病理切片圖(H&E stain) ……………………218
圖六e、CCLS組大白鼠肝臟病理切片圖(H&E stain) …………………219
圖七、長期餵食酒精或注射四氯化碳對於大白鼠肝臟微粒體中細胞色素P450 2E1蛋白質表現之影響……………………………………220


附錄………………………………………………………………………221
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行政院衛生署,國人每年酒類消費量之統計, 2001。
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