臺灣博碩士論文加值系統

先前研究指出，葉酸缺乏會導致動物肝臟粒線體產生大片段DNA斷損。營養性葉酸缺乏或此種粒線體DNA斷損會對粒線體造成何種傷害，則尚未明瞭。本研究以動物模式或初代肝細胞培養模式，探討葉酸缺乏或補充對大白鼠肝臟粒線體基因斷損，功能性及氧化傷害的影響。投予Wistar大白鼠葉酸缺乏或葉酸補充飲食2~4週後，取肝臟粒線體分析粒線體DNA斷損、基因缺失、粒線體脂質過氧化傷害及粒線體蛋白質氧化傷害；或以二階段灌流法取初代肝細胞以螢光染色經細胞流式儀分析胞內活性氧物種、粒線體膜電位、粒線體質量及細胞色素c氧化酶活性。實驗結果顯示，葉酸缺乏組肝臟粒線體確實出現大片段DNA斷損；葉酸補充組則無此斷損。以半定量聚合酶連鎖反應分析位於斷損片段中的COXⅢ基因，葉酸缺乏組肝臟粒線體COXⅢ基因顯著流失，約減少為葉酸補充組的70%。COXⅢ基因轉錄的蛋白質為粒線體呼吸電子傳遞鏈複合體Ⅳ，即細胞色素c氧化酶（cytochrome c oxidase, COX）的重要次單元；可調節複合體Ⅳ的氫質子通道。葉酸缺乏組初代肝細胞COX活性顯著低於葉酸補充組，且其粒線體膜電位降低，去極化細胞百分比及粒線體質量流失情形顯著高於葉酸補充組；額外添加葉酸可顯著恢復初代肝細胞COX酵素活性與顯著改善因金屬離子誘發產生低膜電位細胞的百分比及粒線體質量流失的情形。葉酸缺乏組之肝臟粒線體功能性損傷與氧化壓力的增加具相關性。其初代肝細胞胞內活性氧物種、超氧陰離子含量與肝臟粒線體蛋白質氧化傷害顯著高於葉酸補充組；額外添加葉酸可清除金屬離子誘發葉酸缺乏初代肝細胞胞內產生之超氧陰離子含量，並且有效降低金屬離子誘發的肝臟粒線體脂質過氧化傷害及粒線體蛋白質氧化傷害性。綜合上述結果，我們發現營養性葉酸缺乏會導致肝臟粒線體產生大片段DNA斷損以致粒線體COXⅢ基因流失；其初代肝細胞粒線體COX活性降低、膜電位降低、氧化壓力升高及顯著增加肝臟粒線體蛋白質氧化傷害；補充葉酸則可保護肝臟粒線體基因完整性與功能性，降低內源性或外源性蛋白質氧化傷害。

Previous studies revealed that folate deficiency resulted in large scale deletion of mitochondrial DNA（mtDNA） in rat liver. Effects of folate deficiency on this mtDNA deletion and mitochondrial functions were largely unknown. These questions were studied using animals model as well as primary culture hepatocytes. Liver mitochondria extracted from Wistar rats fed with folate-deficient or folate-supplemented diets were analyzed for common deletion of mtDNA, gene loss and oxidative damage. Primary hepatocytes from those rats were cultured to assess reactive oxygen species（ROS）, mitochondrial membrane poteinal, mitochondrial mass and cytochrome c oxidase activity. Our data showed that folate-deficient rat liver but not folate-supplemented groups exhibited mtDNA common deletion（8103 bp~12936 bp）. Using semiquantitative PCR methods, mitochondrial COXⅢ gene levels in folate-deficient rat liver were significantly decreased to 70% of gene levels in folate-supplemented rat liver. COXⅢ gene encoded on subunit of mitochondrial respiratory complex Ⅳ （cytochrome c oxidase, COX）, which was associated with proton pumping and electron transfer. Primary hepatocytes isolated from folate-deficint rats showed significantly lower levels of enzymatic activity of COX, mitochondrial membrane potenials and mitochondrial mass as compared to those in primary hepatocytes from folate-supplement rats. Supplementation of folate to folate-deficient hepatocyte cultures confer protection against metal ion-induced mitochondrial dysfunction. Mitochondrial dysfunction in folate-deficient hepatocytes were associated with increased intracellular ROS levels, particular for superoxide generation, and oxidative damage to mitochondrial protein . Supplementation of folate to animals or to hepatocytes significantly decreased the severity of oxidative stress（superoxide generation） and prevented from metal ion-induced mitochondrial oxidative damage, particularly for protein oxidative damage. In conclusion, dietary folate deficiency could result in mitochondrial COXⅢ gene loss, mitochondrial dysfunction and increased oxidative damage to mitochondrial protein in rat liver. In vivo and in vitro folate supplementation protected against endogenous or oxidants-induced liver mitochondrial decay and dysfunction.

目錄
頁次
中文摘要……………………………………………………………………i
英文摘要……………………………………………………………….….iii
目錄…...…………………………………………………………………....v
圖目錄…………………………………………………………...…………x
表目錄…………………………………………………………………….xii
第一章 前言…………………………………………………………...…..1
第二章 文獻回顧
一、 葉酸……………………………………………………………….2
（一） 葉酸營養生化代謝…………………………………………2
（二） 葉酸抗氧化特性……………………………………………3
（三） 葉酸營養與氧化傷害………………………………………3
二、 內生性氧化壓力來源：粒線體………………………………….4
（一） 粒線體能量代謝生化功能………………………………....4
（二） 粒線體DNA與呼吸電子傳遞鏈的關係………………….4
（三） 粒線體DNA變異對粒線體生化功能的影響…………….7
（四） 粒線體調節細胞程式凋亡訊息傳遞………………………9
（五） 葉酸與粒線體功能保護性………………………………..10
三、研究目的………………………………………………………….11
第三章 材料與方法
一、 實驗設計………………………………………………………12
二、 實驗材料………………………………………………………12
（一） 實驗動物………………………………………………...12
（二） 飼料……………………………………………………...12
（三） 細胞培養材料…………………………………………...13
（四） 分析用試藥……………………………………………...13
三、 實驗方法………………………………………………………13
（一） 初代肝細胞之分離……………………………………...13
（二） 初代肝細胞培養………………………………………...14
1. 培養皿前處理…………………………………………...14
2. 培養液配製………………………………………….…..14
3. 培養條件………………………………………….……..14
（三） 細胞實驗分析項目……………………………….……..15
1. 細胞色素c氧化酶之染色……………………….……..15
2. 細胞粒線體膜電位之測定……………………….……..15
3. 細胞粒線體質量之測定……………………….………..16
4. 細胞過氧化氫含量測定……………………….………..16
5. 細胞內超氧陰離子含量測定……………………….…..17
（四） 肝臟粒線體之粗萃取……………………….…………..18
（五） 蛋白質含量測定………………………………………...18
（六） 動物實驗分析項目……………………………………...19
1. 粒線體指標酵素活性測定……………………………...19
2. 粒線體脂質過氧化測定………………………………...19
3. 粒線體蛋白質氧化傷害測定…………………………...20
4. 粒線體DNA之分析
（1） Total DNA之萃取………..……………………...21
（2） 雙股DNA聚合酶連鎖反應…………………….22
（3） 粒線體基因定序………………………………...23
（七） 統計分析………………………………………………...26
第四章 結果………………………………………………………….…..27
一、 實驗動物之生長情形………………………………….…27
二、 葉酸營養狀態對老鼠肝臟粒線體DNA斷損的影響…..27
三、 葉酸營養狀態對老鼠肝臟粒線體COXⅢ基因含量之影響………………………………………………………….28
四、 COXⅢ基因之部分定序…………………………….……29
五、 葉酸營養狀態對大白鼠初代肝細胞細胞色素c活性的影響………………………………………………………….29
六、 葉酸營養狀態對大白鼠初代肝細胞粒線體膜電位改變的影響………………………………………………………30
七、 葉酸營養狀態對大白鼠初代肝細胞粒線體膜質量的影響.. ……………………………………………………….31
八、 葉酸營養狀況對大白鼠肝細胞產生氧化壓力的影響…31
九、 補充葉酸對清除金屬離子誘發大白鼠肝細胞產生活性氧物種之影響.. …………………………………………….32
十、 葉酸營養狀況對大白鼠肝臟粒線體脂質過氧化傷害與蛋白質氧化傷害的影響…………………………………....32
十一、 In vitro 補充葉酸在過氧化氫誘發下對大白鼠肝臟粒線體脂質過氧化的影響…………………………………34
十二、 In vitro 補充葉酸在鐵離子誘發下對大白鼠肝臟粒線體脂質過氧化的影響……………………………………34
十三、 In vitro 補充葉酸在鐵離子與過氧化氫誘發下對大白鼠肝臟粒線體脂質過氧化的影響………………………34
十四、 In vitro 補充葉酸在過氧化氫誘發下對大白鼠肝臟粒線體蛋白質氧化損傷的影響……………………………35
十五、 In vitro 補充葉酸在鐵離子誘發下對大白鼠肝臟粒線體蛋白質氧化損傷的影響………………………………36
十六、 In vitro 補充葉酸在鐵離子與過氧化氫誘發下對大白鼠肝臟粒線體蛋白質氧化損傷的影響…………………37
十七、 葉酸補充量對大白鼠肝臟粒線體分離液之脂質過氧化與蛋白質氧化損傷之相關性……………………………38
第五章 討論……………………………………………………………..40
第六章 結論……………………………………………………………..48
第七章 參考文獻………………………………………………………..73
附錄………………………………………………………………………82

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