臺灣博碩士論文加值系統

葉酸是參與同胱胺酸轉換為甲硫胺酸的重要輔因子，因此葉酸缺乏會導致細胞中同胱胺酸的堆積。過去研究顯示，葉酸缺乏會引起Hep G2 cells細胞週期特異性的細胞凋亡，並且指出，葉酸缺乏會透過同胱胺酸的堆積引起H2O2大量產生誘導細胞內氧化壓力，並增加NF-B的活化，使細胞走向凋亡路徑。本研究想更進一步證明，葉酸缺乏是否能透過iNOS表現的增加誘導NO的大量產生使細胞內GSH耗盡，此外，也要證明-catenin的下降是否對NO所誘導的細胞凋亡更敏感。結果發現葉酸缺乏確實會誘導細胞內氧化壓力與硝化壓力的上升，並進一步的利用氧化還原蛋白質體學偵測是否氧化修飾性蛋白質增加於葉酸缺乏的Hep G2細胞，結果指出，有三個carbonylated proteins增加於葉酸缺乏第一週的細胞，包括HSP 60，為粒線體的chaperone protein，可保護粒線體免於氧化壓力的傷害；Protein disulfide isomerase(PDI)為內質網的chaperone，可修復某些氧化態的蛋白質，並影響細胞內鈣離子的平衡；ATP synthetase  chain導致細胞ATP depletion使細胞造成apoptosis。本研究認為葉酸缺乏所引起的細胞凋亡為許多因子所造成，透過Hcy誘導細胞內氧化壓力與硝化壓力的增加，接著造成關鍵性的三個氧化修飾性蛋白質增加，最後都造成葉酸缺乏誘導細胞凋亡的結果。

Folate coenzymes are critical for de novo synthesis of pruine and thymidine, for methionine from homocysteine. Our group has previously reported that folate deficiency could induce a cell cycle-specific apoptosis in Hep G2 cells. Subsequently, we showed folate deficiency-induced oxidative stress and apoptosis was mediated via homocysteine-dependent overproduction of H2O2 and enhanced activation of NF-B. In this study, we further demonstrate that folate deficiency can elicit nitrosative stress by overproducing nitric oxide (NO) mediated via elevated iNOS expression leading to intracellular GSH depletion as reflected by the decreased chloromethyl- fluorescein-diacetate/ GSH fluorescence intensity measured by confocal microscopy. Along the same vein, we also demonstrate that folate deficiency can promote the downregulation of -catenin, a major player in Wnt signaling pathway. This process can also sensitize Hep G2 cells to increased sensitivity to NO-mediated apoptosis. Based on these findings, we conclude that folate deficiency can elicit both oxidative and nitrosative stresses, and thus the proper functions of an array of target proteins can be oxidatively modified by these processes. For this reason, we set out to explore the apoptosis-related target proteins that are being oxidatively modified by the oxidative and nitrosative stresses instigated by folate deficiency in Hep G2 cells using proteomic technique. We found out that three carbonylated proteins were upregulated during an episode of folate deficiency as identified by MALDI-TOF. These proteins included molecular chaperone HSP 60, protein disulfide isomerase (PDI), the mitochondrial complex V subunit, ATP synthetase -chain. Functionally speaking, HSP 60 is a mitochondrial chaperone, and its overexpression can protect mitochondrial function and prevent apoptosis. Thus, its malfunction can render these cells to be susceptible to oxidative stress-mediated apoptosis. Next, PDI an ER chaperone, could interact with damaged target proteins to assist their refolding so that the toxic effective of oxidative stress could be alleviated. The oxidative modification of PDI, the protective function against oxidative stress would be lost. Finally, the oxidative modification of ATP synthetase -chain could also lead to apoptosis due to the depletion of ATP production. Taken together, we suggest that the mechanism for folate deficiency-associated apoptosis is multi-factorials which may initially involve the occurrence of Hcy-mediated oxidative/ nitrosative stresses and consequently resulted in the oxidatively modification of some key proteins, such as HSP 60, PDI and ATP synthetase -chain, via carbonylation reaction. The oxidative modification of these target proteins can ultimately lead to the apoptotic cell death via the drastic changes of their proper conformation caused by oxidative/ nitrosative stresses instigated by folate deficiency.

目 錄
縮寫表…………………………………...………………………………iii
中文摘要…………………………………...……………………………iv
英文摘要………………………………………………………………....v
目錄………………..……………………………………………………vii
第一章 簡介………………………………………………………… 1
1.1 葉酸…………………………………...…...…...……………. 2
1.2 葉酸缺乏與氧化壓力(Oxidative stress)之相關性……..….... 3
1.3 葉酸缺乏與硝化壓力(Nitrosative stress)之相關性……….... 5
1.4 氧化還原蛋白質體學 (Redox proteomics) ...…........………. 7
1.4.1 氧化修飾性蛋白質 (Oxidatively modified proteins) ... 8
1.4.1.1 Carbonylation…….......................….…………. 8
1.4.1.2 Nitration……...................................…………. 10
1.4.1.3 Glutathionylation……..................…...………. 10
1.4.1.4 S-nitrosylation…….........................…………. 10
1.5 葉酸缺乏於蛋白質體學之研究.......................................….. 11
1.6 研究目的(Specific Aims) .....................................…………. 13
第二章 材料與方法……………….....……………………………. 15
2.1 實驗材料……………………………………………...…… 15
2.1.1 細胞株與細胞培養基……………………...…..…… 15
2.2 實驗方法……………………………………………...…… 16
2.2.1 Cell culture………………………………..........…… 16
2.2.2 細胞內ROS的偵測…………………................…… 16
2.2.3 細胞內Nitric Oxide (NO)的偵測………...........…… 16
2.2.4 Mitochondrial membrane potential (Δm)的偵測….. 17
2.2.5 細胞內GSH content的偵測……………….......…… 17
2.2.6 Western blot…………………..............................…… 18
2.2.7 Two-dimensional electrophoresis偵測氧化修飾性蛋白質….............................................................................. 18
2.2.8 Silver stain………………………………............…… 19
2.2.9 In-gel digestion…………………..........…………...… 19
2.2.10 MALDI-TOF質譜分析………………...…......…… 19
2.2.11 測定細胞內ATP之含量………………...........…… 19
2.2.12 Immunofluorescence stain………………….......… 20
第三章 結果………...……………………..………………………. 21
3.1 葉酸缺乏對人類肝癌細胞株Hep G2細胞內ROS產生之影響…………………………………………………………. 21
3.2 葉酸缺乏對人類肝癌細胞株Hep G2細胞Δm之影響… 21
3.3 人類肝癌細胞株Hep G2細胞於葉酸缺乏下造成nitrosative stress之影響……………………...………………………. 23
3.4 葉酸缺乏對人類肝癌細胞株Hep G2細胞內GSH含量之影響…………………………………………………………. 23
3.5 利用DNPH與二維電泳之Western blot分析Hep G2細胞於葉酸缺乏下產生的氧化修飾性蛋白質………………..…. 23
3.6 偵測細胞內ATP的含量…………………….……………. 24
3.7 免疫螢光染色偵測葉酸缺乏細胞內PDI與HSP60蛋白質的表現……………………..………………………………. 24
第四章 討論………...……………………..………………………. 26
4.1 Oxidative stress和Nitrosative stress與葉酸缺乏誘導之cell apoptosis……...……..……………..………………………. 26
4.2 氧化修飾性蛋白質與葉酸缺乏誘導cell apoptosis之相關性……...……………………..……...……………………. 28
4.2.1 ATP synthetase  chain……………………………. 28
4.2.2 Heat shock protein 60………...……………………. 28
4.2.3 Protein disulfide isomerase……..……...………….. 29
第五章 結論………...……………………..………………………. 31
第六章 參考文獻….……………………..………………………... 32
第七章 圖表….………………...…..……………………...……..... 40

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