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研究生:吳禮安
研究生(外文):WU LI AN
論文名稱:活性氧及高粒線體鈣離子堆積在葉酸缺乏情況下誘發之人類葡萄糖六磷酸去氫酶缺乏纖維母細胞程式凋亡所扮演之角色研究
論文名稱(外文):Role of Reactive Oxygen Species (ROS) and Mitochondria Calcium Overloading on the Folic Acid Deprivation-Triggered Apoptosis of Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient Human Foreskin Fibroblasts
指導教授:劉燦榮劉燦榮引用關係
指導教授(外文):LIU TSAN ZON
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:55
中文關鍵詞:葉酸氧化壓力程式凋亡
外文關鍵詞:Folic AcidOxidative stressApoptosis
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葡萄糖六磷酸去氫脢 (Glucose-6-phosphate dehydrogenase, G6PD)主要是催化產生NADPH,來維持細胞內氧化還原反應的平衡。G6PD的缺乏會導致人類纖維母細胞傾向於生長遲緩及加速細胞老化。
此次研究的目的可分為兩點:(1)探索葉酸缺乏引起HFF-1及HFF-3走向凋亡的可能性。此目的是想說明葉酸缺乏導致細胞凋亡的現象與活性氧所引起的粒線體鈣離子過度負荷有關;(2)利用雷射掃瞄共軛焦顯微鏡即時去分析HFF-1及HFF-3在葉酸缺乏早期的反應下谷胱甘肽(glutathione, GSH)的消耗及脂質過氧化的程度。因此,粒線體鈣離子在細胞凋亡的過程中是否扮演著重要的角色,在此次研究中將會被解答。
利用DCF-DA當作螢光探針,首先去偵測HFF-1及HFF-3在葉酸缺乏下粒線體活性氧的產生。結果顯示HFF-1培養於葉酸缺乏環境下七天相較於培養於葉酸健全的環境,其粒線體會產生大量的活性氧,之後,利用CMF-DA及BODIPY當作探針,葉酸缺乏對兩者細胞所造成的早期反應下,會因為葉酸導致的氧化壓力而引起谷胱甘肽的消耗及脂質過氧化。利用Fluo-4(細胞質鈣離子探針)及Rhod-2(粒線體鈣離子探針)當作探針,去偵測鈣離子的分布。在此次研究結果發現只有HFF-1藉由Rhod-2所呈現的螢光的增加可以反映出鈣離子堆積的增加。由這些結果推測,粒線體鈣離子的過度負荷可能會增加HFF1對於葉酸缺乏引起細胞凋亡的敏感性。更進一步地,也發現粒線體膜電位在葉酸缺乏情況下有發生改變。因此,由上述的實驗結果可以推測一個可能的假說,那就是缺乏的纖維母細胞會產生大量的活性氧,而活性氧就會引起谷胱甘肽的消耗、脂質過氧化及脂質過氧化最終會造成膜的傷害。所以,鈣離子迅速進入粒線體的內膜及粒線體膜電位的改變最終導致細胞凋亡的發生。G6PD的缺乏會使纖維母細胞因為雙重氧化壓力下更傾向於因葉酸缺乏導致的凋亡。
Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of NADPH for the proper maintenance of the cellular redox balance, G6PD deficiency predispose human fibroblasts (HFF) to retarded growth and accelerated cellular senescence. Thus far, the pivotal role of a micronutrient, such as folic acid, on the cellular functions of the normal and G6PD-deficient HFF have never been investigated.
The objectives of the present research are two-fold: (1) To explore the possibility that folic acid deficiency can trigger the occurrence of apoptotic cell death in G6PD-deficient as well as G6PD-normal HFFs. Emphasis will be to elucidate if folate deficiency-induced apoptosis involves reactive oxygen species (ROS)-mediated mitochondrial calcium overload pathway; (2) Real time analysis of living intact cell in single cell level to address the questions if glutathione (GSH) depletion and lipid peroxidation are involved in the very early responses of both HFF-1 and HFF-3 fibroblasts to folate deficiency-induced apoptosis using probe-based confocal fluorescence imaging technique. Furthermore, the possibility as to whether or not the mitochondria calcium overload plays a pivotal role in arbitrating apoptotic process will be addressed in this project.
Using DCF-DA as the fluorescence probe, we first monitored the production of mitochondrial ROS (mROS) in both types of fibroblasts cultivated under folate-deficiency condition. Our data indicated that HFF-1 cells (G6PD-deficient cells) produced substantially higher amounts of mROS than their folate-sufficient counterparts (HFF-3 cells) cultivated under folate-deficiency condition seven days. Next, using CMF-DA and BODIPY as the probes, we can able to attest that the folate-deficiency condition (7th day) could elicit GSH depletion and lipid peroxidation in the very early response of both types of fibroblasts to folate-induced oxidative stress situation. Using Fluo-4 (for cytosolic Ca++ probe ) and Rhod-2 (for mitochondrial Ca++ probe) as the probes, we also measured the temporal and spatial distribution of Ca++. In this study, we found that only HFF-1 cells had had increased quantity of Ca++ accumulation as reflected by the increased exhibition of the Rhod-2-mediated orange colored fluorescence. These data attested that mitochondrial calcium overload (mCa++) was the major contributing factor for increased susceptibility of HFF-1 cells to folate deficiency-induced apoptosis as compared to their folate-sufficient counterparts. In parallel, we also found out that increased mCa++ levels correlated with the loss of mitochondrial membrane potential (Δψm). Taken together, our data indicated folate-deficient fibroblasts was mediated via early ROS-evoked GSH depletion and lipid peroxidation and lipid peroxidation resulting in membrane damages. This was followed by a sudden inflex of Ca++ into mitochondria and thus Δψm changed leading to the eventual occurrence of apoptosis. G6PD-deficeincy rendered fibroblasts more prone to folate deficiency-induced apoptosis because of the double stresses situation imposed on these cells.
目錄
指導教授推薦書…………………………………………………………i
口試委員會審定書……………………………………………………...ii
論文著作授權書………………………………………………………..iii
誌謝……………………………………………………………………...iv
中文摘要…………………………………………………………………v
英文摘要………………………………………………………………...vi
目錄……………………………………………………………………..vii
第一章 背景……………………………………………………………1
第二章 材料與方法..............................................................................15
第三章 結果…………………………………………………………..21
第四章 討論…………………………………………………………..29
參考文獻………………………………………………………………..34
第五章 圖與表………………………………………………………..39
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