跳到主要內容

臺灣博碩士論文加值系統

(100.28.2.72) 您好!臺灣時間:2024/06/14 02:30
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:黃奕仁
研究生(外文):Yi Ren Huang
論文名稱:探討還原型穀胱甘肽的再生異常對葡萄糖-6-磷酸脫氫酶缺乏細胞之影響
論文名稱(外文):Ineffective glutathione regeneration on G6PD-knockdown Hep G2 cells
指導教授:趙崇義趙崇義引用關係
指導教授(外文):P. T. Y. Chiu
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:2009
畢業學年度:97
論文頁數:73
中文關鍵詞:G6PDNADPHNAD kinase
相關次數:
  • 被引用被引用:0
  • 點閱點閱:298
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
葡萄糖-6-磷酸脫氫酶 ( G6PD ) 是五碳糖磷酸化途徑 ( Pentose phosphate pathway ) 的第一個酵素,也是速率決定步驟。G6PD藉由調控還原態菸醯胺腺嘌呤二核酸磷酸 ( NADPH ) 的再生,維持細胞內還原態 ( GSH ) 與氧化態的穀胱甘肽 ( GSSG ) 之間的比值。我們利用RNAi的技術建立了G6PD knockdown的HepG2細胞 ( Gi ) 作為細胞模式去闡述在氧化壓力下GSH再生異常造成的補償機制,在正常生長情況下,Gi細胞NADPH / NADP+ and GSH / GSSG比值比對照組細胞 ( Sc ) 相對較低。當接觸到氧化劑diamide時,Gi細胞對於氧化劑的傷害較為敏感,且GSH的再生與GSSG的清除效率較差。另外這兩株細胞在diamide的處理下都呈現NAD+濃度降低與NADP+濃度增加的情況,但是Gi細胞的改變量比起Sc細胞更加顯著。我們還發現Gi細胞在diamide處理下,增強了NAD kinase的活性。雖然在G6PD knockdown細胞有代償性的NADPH濃度增加的情況,但是依然有GSH的再生與GSSG的清除效率較差的情況。因此我們推測G6PD藉由有效的GSH再生達到保護細胞對抗氧化壓力造成的傷害。
Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is important to maintain intracellular reduced and oxidized glutathione ( GSH / GSSG ) ratio via NADPH regeneration. G6PD knockdown HepG2 ( Gi ) cells by RNAi technique were used as a model to elucidate the compensatory mechanism for ineffective GSH regeneration in G6PD knockdown cells under oxidative stress. Under basal condition, intracellular levels of NADPH / NADP+ and GSH / GSSG ratio were lower in Gi cells than those in control ( Sc ) cells. When cells were exposed to diamide, Gi cells were more susceptible to diamide-induced cell death compared with Sc cells. GSH regeneration and GSSG clearance were ineffective in Gi cells. NADP+ level was increased and NAD+ level was decreased in both cells. However, the increase of NADP+ and the decrease of NAD+ were larger in Gi cells than those in Sc cells. We found that NAD kinase activity was increased in Gi cells more dramatically than Sc cells when cells were exposed to diamide. Although NADPH level was significantly increased in G6PD knockdown cells, which were with an impaired ability to regenerate glutathione after diamide treatment. Our findings suggest that G6PD confers protection against oxidant-induced cytotoxicity through effective glutathione regeneration.
目錄
指導教授推薦書………………………………………………………
論文口試委員會審定書………………………………………………
長庚大學授權書………………………………………………………iii
誌謝……………………………………………………………………iv
中文摘要………………………………………………………………v
英文摘要………………………………………………………………vi
目錄……………………………………………………………………vii
圖表目錄………………………………………………………………viii
前言……………………………………………………………………01
實驗目的………………………………………………………………07
實驗設計………………………………………………………………08
實驗材料與方法………………………………………………………09
實驗結果………………………………………………………………29
討論……………………………………………………………………35
參考文獻………………………………………………………………40
圖表……………………………………………………………………50
附錄……………………………………………………………………62


圖表目錄
圖一、實驗設計………………………………………………………50
圖二、確認G6PD knockdown細胞株之建立………………………51
圖三、G6PD knockdown HepG2細胞對於氧化劑diamide之敏感性大於對照細胞…………………………………………………52
圖四、G6PD knockdown Hep G2細胞在diamide處理下,GSH的回復速率與GSSG的清除速率都顯著較慢……………………53
圖五、G6PD knockdown細胞在diamide處理下其細胞內NADP+與NADPH含量顯著增加………………………………………54
圖六、G6PD knockdown細胞在diamide處理下其NAD kinase活性顯著增加………………..………………………………………56
圖七、G6PD knockdown細胞在diamide處理下,其isocitrate dehydrogenase和malic enzyme活性並無顯著改變……57
圖八、G6PD knockdown細胞在diamide處理下glutathione reductase活性並無顯著改變......................................................58
圖九、 G6PD knockdown細胞在diamide處理下其蛋白glutathionylation程度顯著增加...............................................59
圖十、根據實驗結果推論之可能機制圖…………………………61
[1] Kumar, B.; Koul, S.; Khandrika, L.; Meacham, R. B.; Koul, H. K. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res 68:1777-1785; 2008.
[2] Bondarenko, O. I.; Sahach, V. F. [Role of mitochondria in reglulation of endothelial cell hyperpolarization to acetylcholine]. Fiziol Zh 52:6-11; 2006.
[3] Jezek, P.; Hlavata, L. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int J Biochem Cell Biol 37:2478-2503; 2005.
[4] Schafer, M.; Werner, S. Oxidative stress in normal and impaired wound repair. Pharmacol Res 58:165-171; 2008.
[5] Santangelo, F.; Witko-Sarsat, V.; Drueke, T.; Descamps-Latscha, B. Restoring glutathione as a therapeutic strategy in chronic kidney disease. Nephrol Dial Transplant 19:1951-1955; 2004.
[6] Meister, A. Glutathione metabolism and its selective modification. J Biol Chem 263:17205-17208; 1988.
[7] Deneke, S. M.; Fanburg, B. L. Regulation of cellular glutathione. Am J Physiol 257:L163-173; 1989.
[8] Wu, D.; Meydani, S. N.; Sastre, J.; Hayek, M.; Meydani, M. In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J Nutr 124:655-663; 1994.
[9] Zinellu, A.; Sotgia, S.; Usai, M. F.; Chessa, R.; Deiana, L.; Carru, C. Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection. Electrophoresis 26:1963-1968; 2005.
[10] Anderson, M. E. Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 111-112:1-14; 1998.
[11] Hayes, J. D.; McLellan, L. I. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 31:273-300; 1999.
[12] Carlucci, F.; Tabucchi, A.; Biagioli, B.; Sani, G.; Lisi, G.; Maccherini, M.; Rosi, F.; Marinello, E. Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione. Electrophoresis 21:1552-1557; 2000.
[13] Meister, A.; Anderson, M. E. Glutathione. Annu Rev Biochem 52:711-760; 1983.
[14] Misra, I.; Griffith, O. W. Expression and purification of human gamma-glutamylcysteine synthetase. Protein Expr Purif 13:268-276; 1998.
[15] White, A. T.; Spence, F. J.; Chipman, J. K. Glutathione depletion modulates gene expression in HepG2 cells via activation of protein kinase C alpha. Toxicology 216:168-180; 2005.
[16] Winterbourn, C. C.; Metodiewa, D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys 314:284-290; 1994.
[17] Dickinson, D. A.; Forman, H. J. Glutathione in defense and signaling: lessons from a small thiol. Ann N Y Acad Sci 973:488-504; 2002.
[18] Bermejo, P.; Martin-Aragon, S.; Benedi, J.; Susin, C.; Felici, E.; Gil, P.; Ribera, J. M.; Villar, A. M. Peripheral levels of glutathione and protein oxidation as markers in the development of Alzheimer's disease from Mild Cognitive Impairment. Free Radic Res 42:162-170; 2008.
[19] Jones, D. P. Extracellular redox state: refining the definition of oxidative stress in aging. Rejuvenation Res 9:169-181; 2006.
[20] Brioukhanov, A. L.; Netrusov, A. I. Catalase and superoxide dismutase: distribution, properties, and physiological role in cells of strict anaerobes. Biochemistry (Mosc) 69:949-962; 2004.
[21] Scott, M. D.; Zuo, L.; Lubin, B. H.; Chiu, D. T. NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes. Blood 77:2059-2064; 1991.
[22] Ho, H. Y.; Cheng, M. L.; Cheng, P. F.; Chiu, D. T. Low oxygen tension alleviates oxidative damage and delays cellular senescence in G6PD-deficient cells. Free Radic Res 41:571-579; 2007.
[23] Wan, G. H.; Lin, K. K.; Tsai, S. C.; Chiu, D. T. Decreased glucose-6-phosphate-dehydrogenase (G6PD) activity and risk of senile cataract in Taiwan. Ophthalmic Epidemiol 13:109-114; 2006.
[24] Ho, H. Y.; Wei, T. T.; Cheng, M. L.; Chiu, D. T. Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status. J Agric Food Chem 54:1638-1645; 2006.
[25] Ho, H. Y.; Cheng, M. L.; Chiu, D. T. G6PD--an old bottle with new wine. Chang Gung Med J 28:606-612; 2005.
[26] Ho, H. Y.; Cheng, M. L.; Lu, F. J.; Chou, Y. H.; Stern, A.; Liang, C. M.; Chiu, D. T. Enhanced oxidative stress and accelerated cellular senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts. Free Radic Biol Med 29:156-169; 2000.
[27] Cheng, M. L.; Ho, H. Y.; Liang, C. M.; Chou, Y. H.; Stern, A.; Lu, F. J.; Chiu, D. T. Cellular glucose-6-phosphate dehydrogenase (G6PD) status modulates the effects of nitric oxide (NO) on human foreskin fibroblasts. FEBS Lett 475:257-262; 2000.
[28] van der Donk, W. A.; Zhao, H. Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol 14:421-426; 2003.
[29] Pollak, N.; Dolle, C.; Ziegler, M. The power to reduce: pyridine nucleotides--small molecules with a multitude of functions. Biochem J 402:205-218; 2007.
[30] Liguzinski, P.; Korzeniewski, B. How to keep glycolytic metabolite concentrations constant when ATP/ADP and NADH/NAD+ change. Syst Biol (Stevenage) 153:332-334; 2006.
[31] Eto, K.; Tsubamoto, Y.; Terauchi, Y.; Sugiyama, T.; Kishimoto, T.; Takahashi, N.; Yamauchi, N.; Kubota, N.; Murayama, S.; Aizawa, T.; Akanuma, Y.; Aizawa, S.; Kasai, H.; Yazaki, Y.; Kadowaki, T. Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion. Science 283:981-985; 1999.
[32] Biaglow, J. E.; Miller, R. A. The thioredoxin reductase/thioredoxin system: novel redox targets for cancer therapy. Cancer Biol Ther 4:6-13; 2005.
[33] Kalinina, E. V.; Chernov, N. N.; Saprin, A. N. Involvement of thio-, peroxi-, and glutaredoxins in cellular redox-dependent processes. Biochemistry (Mosc) 73:1493-1510; 2008.
[34] Kawai, S.; Fukuda, C.; Mukai, T.; Murata, K. MJ0917 in archaeon Methanococcus jannaschii is a novel NADP phosphatase/NAD kinase. J Biol Chem 280:39200-39207; 2005.
[35] Lerner, F.; Niere, M.; Ludwig, A.; Ziegler, M. Structural and functional characterization of human NAD kinase. Biochem Biophys Res Commun 288:69-74; 2001.
[36] Hamel, R.; Appanna, V. D.; Viswanatha, T.; Puiseux-Dao, S. Overexpression of isocitrate lyase is an important strategy in the survival of Pseudomonas fluorescens exposed to aluminum. Biochem Biophys Res Commun 317:1189-1194; 2004.
[37] Middaugh, J.; Hamel, R.; Jean-Baptiste, G.; Beriault, R.; Chenier, D.; Appanna, V. D. Aluminum triggers decreased aconitase activity via Fe-S cluster disruption and the overexpression of isocitrate dehydrogenase and isocitrate lyase: a metabolic network mediating cellular survival. J Biol Chem 280:3159-3165; 2005.
[38] Beriault, R.; Chenier, D.; Singh, R.; Middaugh, J.; Mailloux, R.; Appanna, V. Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis. Electrophoresis 26:2892-2897; 2005.
[39] Ochiai, A.; Mori, S.; Kawai, S.; Murata, K. Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1. Protein Expr Purif 36:124-130; 2004.
[40] Grose, J. H.; Joss, L.; Velick, S. F.; Roth, J. R. Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress. Proc Natl Acad Sci U S A 103:7601-7606; 2006.
[41] Pollak, N.; Niere, M.; Ziegler, M. NAD kinase levels control the NADPH concentration in human cells. J Biol Chem 282:33562-33571; 2007.
[42] Tien Kuo, M.; Savaraj, N. Roles of reactive oxygen species in hepatocarcinogenesis and drug resistance gene expression in liver cancers. Mol Carcinog 45:701-709; 2006.
[43] Moriya, K.; Nakagawa, K.; Santa, T.; Shintani, Y.; Fujie, H.; Miyoshi, H.; Tsutsumi, T.; Miyazawa, T.; Ishibashi, K.; Horie, T.; Imai, K.; Todoroki, T.; Kimura, S.; Koike, K. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res 61:4365-4370; 2001.
[44] Shen, H. M.; Shi, C. Y.; Shen, Y.; Ong, C. N. Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1. Free Radic Biol Med 21:139-146; 1996.
[45] Youssef, J. A.; Badr, M. Z. Aging and enhanced hepatocarcinogenicity by peroxisome proliferator-activated receptor alpha agonists. Ageing Res Rev 4:103-118; 2005.
[46] Reddy, J. K.; Rao, S.; Moody, D. E. Hepatocellular carcinomas in acatalasemic mice treated with nafenopin, a hypolipidemic peroxisome proliferator. Cancer Res 36:1211-1217; 1976.
[47] Furukawa, O.; Matsui, H.; Suzuki, N.; Okabe, S. Epidermal growth factor protects rat epithelial cells against acid-induced damage through the activation of Na+/H+ exchangers. J Pharmacol Exp Ther 288:620-626; 1999.
[48] Bailey, S. M. A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction. Free Radic Res 37:585-596; 2003.
[49] Wheeler, M. D. Endotoxin and Kupffer cell activation in alcoholic liver disease. Alcohol Res Health 27:300-306; 2003.
[50] Haga, S.; Terui, K.; Fukai, M.; Oikawa, Y.; Irani, K.; Furukawa, H.; Todo, S.; Ozaki, M. Preventing hypoxia/reoxygenation damage to hepatocytes by p66(shc) ablation: up-regulation of anti-oxidant and anti-apoptotic proteins. J Hepatol 48:422-432; 2008.
[51] St-Pierre, J.; Drori, S.; Uldry, M.; Silvaggi, J. M.; Rhee, J.; Jager, S.; Handschin, C.; Zheng, K.; Lin, J.; Yang, W.; Simon, D. K.; Bachoo, R.; Spiegelman, B. M. Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127:397-408; 2006.
[52] Macip, S.; Kosoy, A.; Lee, S. W.; O'Connell, M. J.; Aaronson, S. A. Oxidative stress induces a prolonged but reversible arrest in p53-null cancer cells, involving a Chk1-dependent G2 checkpoint. Oncogene 25:6037-6047; 2006.
[53] Miles, G. P.; Samuel, M. A.; Zhang, Y.; Ellis, B. E. RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3. Environ Pollut 138:230-237; 2005.
[54] Wong, H. L.; Sakamoto, T.; Kawasaki, T.; Umemura, K.; Shimamoto, K. Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice. Plant Physiol 135:1447-1456; 2004.
[55] Blander, G.; de Oliveira, R. M.; Conboy, C. M.; Haigis, M.; Guarente, L. Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. J Biol Chem 278:38966-38969; 2003.
[56] Gao, L. P.; Cheng, M. L.; Chou, H. J.; Yang, Y. H.; Ho, H. Y.; Chiu, D.T.Y. Ineffective GSH regeneration enhances G6PD-knockdown Hep G2 cell sensitivity to diamide-induced oxidative damage. Free Radic Biol Med; 2009.
[57] Coolen, E. J.; Arts, I. C.; Swennen, E. L.; Bast, A.; Stuart, M. A.; Dagnelie, P. C. Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection. J Chromatogr B Analyt Technol Biomed Life Sci 864:43-51; 2008.
[58] Lazzarino, G.; Amorini, A. M.; Fazzina, G.; Vagnozzi, R.; Signoretti, S.; Donzelli, S.; Di Stasio, E.; Giardina, B.; Tavazzi, B. Single-sample preparation for simultaneous cellular redox and energy state determination. Anal Biochem 322:51-59; 2003.
[59] Shin, S. W.; Oh, C. J.; Kil, I. S.; Park, J. W. Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity. Free Radic Res 43:409-416; 2009.
[60] Yang, E. S.; Lee, J. H.; Park, J. W. Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase. Biochimie 90:1316-1324; 2008.
[61] Kosower, N. S.; Kosower, E. M. Diamide: an oxidant probe for thiols. Methods Enzymol 251:123-133; 1995.
[62] Filosa, S.; Fico, A.; Paglialunga, F.; Balestrieri, M.; Crooke, A.; Verde, P.; Abrescia, P.; Bautista, J. M.; Martini, G. Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress. Biochem J 370:935-943; 2003.
[63] Mori, S.; Kawai, S.; Mikami, B.; Murata, K. Crystallization and preliminary X-ray analysis of NAD kinase from Mycobacterium tuberculosis H37Rv. Acta Crystallogr D Biol Crystallogr 57:1319-1320; 2001.
[64] Bieganowski, P.; Seidle, H. F.; Wojcik, M.; Brenner, C. Synthetic lethal and biochemical analyses of NAD and NADH kinases in Saccharomyces cerevisiae establish separation of cellular functions. J Biol Chem 281:22439-22445; 2006.
[65] Magni, G.; Orsomando, G.; Raffaelli, N. Structural and functional properties of NAD kinase, a key enzyme in NADP biosynthesis. Mini Rev Med Chem 6:739-746; 2006.
[66] Berger, F.; Ramirez-Hernandez, M. H.; Ziegler, M. The new life of a centenarian: signalling functions of NAD(P). Trends Biochem Sci 29:111-118; 2004.
[67] Frederiks, W. M.; Vizan, P.; Bosch, K. S.; Vreeling-Sindelarova, H.; Boren, J.; Cascante, M. Elevated activity of the oxidative and non-oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver. Int J Exp Pathol 89:232-240; 2008.
[68] Ayene, I. S.; Biaglow, J. E.; Kachur, A. V.; Stamato, T. D.; Koch, C. J. Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: mechanism and implication. J Cell Biochem 103:123-135; 2008.
[69] Biaglow, J. E.; Ayene, I. S.; Koch, C. J.; Donahue, J.; Stamato, T. D.; Tuttle, S. W. G6PD deficient cells and the bioreduction of disulfides: effects of DHEA, GSH depletion and phenylarsine oxide. Biochem Biophys Res Commun 273:846-852; 2000.
[70] Klatt, P.; Molina, E. P.; De Lacoba, M. G.; Padilla, C. A.; Martinez-Galesteo, E.; Barcena, J. A.; Lamas, S. Redox regulation of c-Jun DNA binding by reversible S-glutathiolation. FASEB J 13:1481-1490; 1999.
[71] Pineda-Molina, E.; Klatt, P.; Vazquez, J.; Marina, A.; Garcia de Lacoba, M.; Perez-Sala, D.; Lamas, S. Glutathionylation of the p50 subunit of NF-kappaB: a mechanism for redox-induced inhibition of DNA binding. Biochemistry 40:14134-14142; 2001.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top