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Author:余朱青
Author (Eng.):YU CHU CHING
Title:葉酸調節肝臟粒線體呼吸酵素複體IV功能性及其機制探討
Title (Eng.):Molecular mechanisms of folate in the regulation of mitochondrial complex IV activity
Advisor:許瑞芬許瑞芬 author reflink
degree:Master
Institution:輔仁大學
Department:食品營養學系
Narrow Field:醫藥衛生學門
Detailed Field:營養學類
Types of papers:Academic thesis/ dissertation
Publication Year:2006
Graduated Academic Year:94
language:Chinese
keyword (chi): 葉酸 呼吸酵素複體IV 氧化性鹼基突變 大片段斷損 基因表現
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粒線體呼吸酵素複體IV (Cytochrome c oxidase;COX)是電子傳遞最終催化中心,是由許多次單元所構成,調節粒線體膜電位及細胞程式凋亡,並與老化及粒線體疾病等氧化壓力相關。葉酸缺乏導致氧化壓力是否直接影響COX活性及其調節機制目前未明。本研究以細胞培養及動物模式,探討葉酸營養對大鼠肝臟粒線體氧化壓力、COX活性及其調節機制。將大鼠肝癌細胞株( H-4-II-E )培養於葉酸缺乏培養液,以流式細胞儀分析顯示胞內活性氧物種顯著升高,COX活性降低。補充葉酸降低胞內氧化壓力及回復COX活性。投予Wistar大鼠葉酸缺乏( 0 mg/kg diet)飲食,收集肝臟粒線體評估氧化壓力,粒線體麩胱苷肽過氧化酶顯著低於對照組。本研究進一步探討葉酸對COX活性之調節機制,葉酸缺乏是否造成COX基因氧化性鹼基突變。主導COX催化功能性的三個次單元COX I,COX II及COX III是由粒線體DNA轉錄,基因定序結果顯示葉酸缺乏並無在此三個次單元造成T→C或G→A氧化性鹼基突變。粒線體DNA大片段斷損與氧化壓力有關,其中COX III基因座落此斷損中,斷損比例會影響COX III基因含量。利用即時定量聚合酶連鎖反應發現葉酸在氧化壓力下不影響粒線體DNA斷損程度,推測COX活性可能不受大片段斷損的影響。利用即時聚合酶連鎖反應SYBR Green I螢光標定方式偵測COX次單元基因表現,葉酸缺乏向上調節COX III、COX VI、COX V、cytochrome c與粒線體DNA複製因子NRF-1 ( nuclear respiratory factor 1 )、mtTFA ( mitochondria transcription factor )、mtSSB ( mitochondria DNA binding protein )、Polγ( DNA polymeraseγ)等基因表現,但粒線體呼吸酵素複體次單元COX IV蛋白質則不受影響。綜合上述,葉酸降低COX活性的機制,不在調節氧化性鹼基突變、DNA大片段斷損、基因及蛋白質表達,顯示可能是由葉酸缺乏導致氧化壓力直接影響COX酵素催化功能性。
目錄
頁次
中文摘要……………………………………………………………………i
英文摘要……………………………………………………………….….iii
縮寫表…...………………………………………………………………....v
圖目錄…………………………………………………………...………...xi
表目錄…………………………………………………………………....xiii
第一章 前言…………………………………………………………...…..1
第二章 文獻回顧………………………………………………………….2
一、葉酸………………………………………………………………….2
(一)葉酸營養生化代謝…………………………….………………2
(二)葉酸的抗氧化特性…………………………………..…..….…3
(三)葉酸營養誘發氧化壓力與細胞程式凋亡………………….…3
二、氧化壓力與粒線體的功能性 …………………………….….….5
(一)氧化壓力與粒線體抗氧化酵素系統……………………….....5
(二)氧化壓力與粒線體DNA損傷………….…………………….7
(三)氧化壓力與粒線體生合成…………………………………….8


三、粒線體呼吸酵素複體IV (cytochrome c oxidase; COX)……......10
(一) COX功能性…………………………………………..……..10
(二)氧化壓力與COX活性……………………………………….11
(三) COX與疾病相關性…………………………………………12
四、葉酸營養不良對粒線體傷害之影響……………………………12
五、研究動機…………………………………………………………14
六、研究目的………………………………………………………….14
第三章 材料與方法
一、實驗材料……………………………………………………...…16
(一)實驗動物…………………….……………………………....16
(二)動物飼料………………….…………………………….…...16
(三)細胞株…………..…………………………………………...17
(四)細胞培養材料……..………………………………………...17
二、實驗方法…………………………………………………………18
(一)初代肝細胞之分離………..………………………………...18
(二)初代肝細胞培養……..……………………………………...19
(三)大白鼠肝癌細胞株H4IIE培養……………………………..19
(四)初代肝細胞粒線體DNA之分析…………………….……..20
1. Total DNA 萃取………….……………………….……..20
2. DNA電泳……………………..………………….……..20
3. 聚合酶連鎖反應……………………………….………..21
4. 即時定量聚合酶連鎖反應…………………….………..22
5. 粒線體基因定序…………………………………….…..23
(五)大白鼠肝癌細胞H4IIE分析項目取..………….…………..27
1. 細胞色素c氧化酶之染色……………………………....27
2. 細胞內過氧化氫含量測定……………………………...27
3. 細胞內超氧陰離子含量測定…………………………...28
(六)肝臟粒線體之粗萃取……………………………………....29
(七)蛋白質含量測定……………………………………………29
(八)粒線體抗氧化酵素系統之測定……………………………30
1. 細胞質液指標酵素活性測定……………………………30
2. 粒線體指標酵素活性測定………………………………30
3. 超氧化歧化酶活性測定…………………………………31
4. 過氧化氫酶活性測定……………………………………32
5. 麩胱苷肽過氧化酶活性測定……………………………32
6. 麩胱苷肽及氧化型麩胱苷肽含量測定…………………33
(九)肝組織mRNA表現量測定…………………………………34
1. Total RNA之萃取………………………………………….34
2. 反轉錄聚合酶鏈鎖反應…………………………………..34
(十)肝組織蛋白質表現量測定…………………………………36
(十一)統計分析….……………………………………………...37
第四章 結果………………………………………………………….…..38
一、葉酸營養狀態與氧化壓力對大白鼠肝臟粒線體COX I、II、III基因突變性之影響………………………………………….…38
二、氧化壓力對葉酸缺乏老鼠肝臟粒線體DNA大片段斷損及COX III Copy number之影響…………………………………….…..38
三、葉酸缺乏對大白鼠肝臟粒線體COX次單元及部份粒線體基因轉錄mRNA表現量之影響….………………..……………….40
四、葉酸缺乏對粒線體生合成轉錄因子之調節.…………………41
五、葉酸營養狀況對老鼠肝臟粒線體抗氧化酵素之影響……….41
六、葉酸營養狀況對大白鼠肝臟粒線體COX IV次單元蛋白質表現量之影響………………………….…………………………42

建立因應氧化壓力調節COX活性的葉酸缺乏肝細胞株模式………..43
一、葉酸營養狀況對H4IIE大白鼠肝癌細胞株生長情形之影響....43
二、有無外源性氧化壓力下之葉酸營養狀況對大白鼠肝癌細胞株H4IIE胞內過氧化氫及超氧陰離子含量影響……..………….43
三、葉酸缺乏大白鼠肝癌細胞株H4IIE在有無外源性氧化壓力下其胞內過氧化氫及超氧陰離子含量影響…………………….44
四、葉酸營養狀況對大白鼠初代肝細胞細胞色素c氧化酶活性之影響…………………………………………………………….45
第五章 討論……………………………………………………………..46
第六章 結論……………………………………………………………..55
第七章 參考文獻………………………………………………………..74
附錄………………………………………………………………………82

圖目錄
頁次
圖一、葉酸營養狀態對大白鼠肝臟粒線體COX I基因序列之影響...…58
圖二、葉酸營養狀態對大白鼠肝臟粒線體COX II基因序列之影響….59
圖三、葉酸營養狀態對大白鼠肝臟粒線體COX III基因序列之影響…60
圖四、葉酸缺乏其老鼠肝臟粒線體COX III與ND1基因之相對含量...61
圖五、銅離子誘發下對葉酸營養狀況與老鼠肝臟粒線體DNA大片段斷損之影響…………………………………………………………...62
圖六、銅離子誘發下葉酸缺乏對老鼠肝臟粒線體DNA大片段斷損之影響…………………………………………………………….……..63
圖七、葉酸缺乏對大白鼠肝臟粒線體COX次單元及部份粒線體基因轉錄mRNA表現量之影響………………………………………...64
圖八、葉酸缺乏對大白鼠肝臟粒線體轉錄因子表現量之影響……….65
圖九、葉酸缺乏對大白鼠肝臟粒線體COX IV蛋白質表現之影響…..67
圖十、H4IIE大白鼠肝癌細胞株培養於葉酸正常、葉酸缺乏培養液及葉酸缺乏培養一週後之細胞生長情形…………………………...…68
圖十一、葉酸補充與t-BH誘發對大白鼠肝癌細胞株H4IIE胞內過氧化氫含量影響……………………………………………………….69
圖十二、葉酸補充與t-BH誘發對大白鼠肝癌細胞株H4IIE胞內超氧陰離子含量影響…………………………………...…….………….70
圖十三、葉酸缺乏一週之大白鼠肝癌細胞株H4IIE其胞內活性氧物種之影響…………………………………………………………….71
圖十四、葉酸缺乏大白鼠肝癌細胞株H4IIE在t-BH氧化壓力下其胞內活性氧物種含量影響……………………………..……………...72
圖十五、葉酸營養狀況對大白鼠初代肝細胞細胞色素c氧化酶活性之影響…………………………………………………………..…...73

表目錄
頁次
表一、本實驗設計所使用之引子與探針序列及其相對應位置……..…56
表二、反轉錄聚合酶鏈鎖反應所使用之引子序列及其相對應位置…. 57
表三、葉酸缺乏對大白鼠肝臟粒線體抗氧化酵素之影響..................….66
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