臺灣博碩士論文加值系統

葡萄糖六磷酸去氫酶Glucose-6-phosphate dehydrogenase (G6PD)是細胞內產生NADPH並維持氧化壓力平衡的重要酵素。雖然G6PD缺乏症是全世界最常見的酵素缺乏症，但我們對於G6PD缺乏會造成紅血球外其他細胞什麼影響目前所知仍然有限。先前我們實驗室曾指出在G6PD缺乏的人類包皮纖維母細胞容易因氧化壓力而導致凋亡。有趣的是，在G6PD缺乏的人類包皮纖維母細胞其Bcl-2蛋白量遠低於G6PD正常的人類包皮纖維母細胞。為了進一步探討G6PD的活性和Bcl-2蛋白量之間的相關性，我們收集了G6PD缺乏的人類嗜中性球和實驗室利用RNAi技術得到的G6PD-knockdown人類肺癌細胞A549、人類肝癌細胞HepG2以及人類血癌細胞HL-60並測其Bcl-2的量。我們發現在G6PD缺乏的人類嗜中性球中與G6PD正常的人類嗜中性球相比會有較低量的Bcl-2蛋白，可是在其他G6PD-knockdown的細胞株中則沒有明顯的差異。儘管G6PD其蛋白量與Bcl-2蛋白量之間有正相關 (R=0.96) 但其RNA卻沒有明顯的差異。而且G6PD缺乏的嗜中性球產生較多量的超氧陰離子 (superoxide, O2-.) 。此外，當受到外來的刺激時 (TNF-alpha) G6PD缺乏的嗜中性球會比較容易凋亡。總結這些數據，我們發現G6PD缺乏的嗜中性球會使得細胞內氧化壓力不平衡並導致訊息傳遞發生改變使得Bcl-2蛋白量下降。而G6PD的活性是如何影響Bcl-2蛋白量這相關的機制便是本實驗室日後的研究目標之一。

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in regulating cellular redox status by generating NADPH. Although its deficiency is the most common enzymopathy affecting over 400 million people world-wide, very little is known about how G6PD-deficiency may affect cells other than red cell. We have previously reported that human G6PD-deficient foreskin fibroblast cells (HFFs) were highly susceptible to oxidant-induced apoptosis. Interestingly, the protein level of Bcl-2 in G6PD-deficient HFFs was much lower than that in normal HFFs. To further investigate the effect of G6PD activity on Bcl-2, we correlate the relationship between G6PD status and Bcl-2 protein/RNA levels in human neutrophils as well as in several cell line with or without G6PD knockdown. Low Bcl-2 protein level was found in G6PD-deficient neutrophils. However, there was no significant change of Bcl-2 protein level in G6PD-knockdown cell lines, such as A549, HepG2 and HL-60 as compared to the control counter parts. There was no difference in RNA levels, albeit a positive correlation (R=0.96) was shown between G6PD and Bcl-2 protein in neutrophils. In response to TNF-alpha, G6PD-deficient neutrophils were more susceptible to apoptosis than normal control and such enhanced susceptibility to oxidant-induced apoptosis can be attributed to the decreased protein level of Bcl-2 in these cells. In addition, G6PD-deficient neutrophils exhibited higher level of superoxide anion than normal neutrophils. Taken together, our findings suggest that G6PD-deficient cells with increased intracellular oxidative stress and altered redox signaling are closely related to Bcl-2 protein level in human neutrophils and primary fibroblasts. The underlying mechanism of how does G6PD status affect Bcl-2 protein level is currently being investigated.

指導教授推薦書
口試委員會審定書
授權書
誌謝 iv
中文摘要 v
英文摘要 vii
目錄 ix
附錄圖表 xiii
第一章 前言 1
1.1 Glucose-6-phosphate dehydrogenase (G6PD)缺乏症與氧化壓 力 1
1.2 氧化壓力和細胞凋亡 3
1.3 Bcl-2家族蛋白 4
1.4蛋白降解作用 5
1.5 ROS和Bcl-2 6
第二章 研究目的 9
第三章 實驗流程 10
第四章 實驗材料及方法 11
4.1實驗方法 11
4.1.1收集G6PD正常與缺乏者全血並分離出嗜中性球 11
4.1.2嗜中性球蛋白質萃取與定量 12
4.1.3利用TNF-α處理人類嗜中性球 13
4.1.4細胞培養 13
4.1.5 G6PD酵素活性測試 13
4.1.6 西方墨點法 14
4.1.7 測量Caspas-3活性 15
4.1.8 測量血球細胞產生超氧陰離子之含量 15
4.1.9利用proteasome抑制劑 (Lactacystin/MG132) 處理人類嗜中性 球 16
4.1.10 全血RNA萃取及濃度測定 16
4.1.11 反轉錄聚合連酶連鎖反應 17
4.1.12 即時定量聚合酶連鎖反應 18
4.1.13 統計分析方法 18
第五章 實驗結果 19
5.1 G6PD 缺乏的人在全血及嗜中性球其G6PD活性皆明顯低於G6PD正常的人 19
5.2 G6PD 缺乏的嗜中性球其Bcl-2的蛋白量明顯低於G6PD正常嗜中性球 20
5.3 人類嗜中性球之G6PD蛋白/活性與Bcl-2蛋白量之間呈正相關 20
5.4 利用辨認不同epitope的Bcl-2抗體去確認Bcl-2蛋白在G6PD缺乏的嗜中性球蛋白量確實低於G6PD正常的嗜中性球 21
5.5 Bcl-2蛋白家族中的其他成員，在G6PD正常與缺乏的嗜中性球中沒有顯著差異 22
5.6 Bcl-2在G6PD缺乏的原代培養細胞中蛋白量會下降，在G6PD-knockdown的細胞株中則沒有這種現象發生 22
5.7 在G6PD缺乏的人類嗜中性球及G6PD–knowdown HL-60 (Neutrophil-like cell) IDH的表現量與正常組相比沒有顯著差異 23
5.8 利用TNF-α在不同時間點處理G6PD正常與缺乏的嗜中性球，發現G6PD缺乏的嗜中性球Caspase-3蛋白量與活性都比G6PD正常的嗜中性球高 23
5.9 G6PD缺乏的嗜中性球超氧陰子的產量比G6PD正常的嗜中性球高 24
5.10 在G6PD正常與缺乏的人類嗜中性球中，Bcl-2的RNA沒有差異 25
5.11 在G6PD正常與缺乏人類嗜中性球中，蛋白的ubiquitination在一般情況下沒有差異 25
5.12 利用proteasome抑制劑(Lactacystin)處理人類嗜中性球後，G6PD缺乏的嗜中性球其Bcl-2不會因proteasome抑制劑(Lactacystin)的處理而有回復的現象 26
5.13 利用proteasome抑制劑(MG132)處理人類嗜中性球後，G6PD缺乏的嗜中性球其Bcl-2不會因proteasome抑制劑 (MG132) 的處理而有回復的現象 26
第六章 討論 28
圖表 34
參考文獻 51


附錄圖表

圖 一、實驗設計流程 34
圖 二、G6PD缺乏者的G6PD活性在全血及人類嗜中性球中明顯比G6PD正常者低 36
圖 三、G6PD缺乏的人類嗜中性球中有較少量的抗凋亡蛋白Bcl-2與較高的凋亡指數(BAX/Bcl-2) 37
圖 四、人類嗜中性球之G6PD蛋白/活性與Bcl-2蛋白量之間呈正相關 39
圖 五、利用不同epitope的抗體去證實G6PD缺乏的嗜中性球的Bcl-2蛋白量確實低於G6PD正常的嗜中性球 40
圖 六、Bcl-2蛋白家族中的其他成員，在G6PD正常與缺乏的嗜中性球中沒有顯著差異 41
圖 七、G6PD缺乏的原代纖維母細胞其Bcl-2蛋白量低於正常對照，而G6PD-knockdown之A549、 HepG2以及HL-60細胞其Bcl-2量與正常對照比較並沒有明顯差異 42
圖 八、在人類嗜中性球或HL-60 (Neutrophil-like cell) 中，IDH (Isocitrate dehydrogenase)表現量沒有顯著差異 43
圖 九、凋亡相關蛋白Caspase-3的蛋白量與活性在G6PD缺乏的嗜中性球中高於G6PD正常的嗜中性球 44
圖 十、G6PD缺乏的人類嗜中性球其超氧陰離子的產量遠高於G6PD正常的人類嗜中性球 46
圖 十一、在G6PD正常與缺乏的人類嗜中性球中，Bcl-2的RNA量沒有顯著差異 47
圖 十二、在G6PD正常與缺乏人類嗜中性球中，全部蛋白的ubiquitination在正常情況下沒有差異 48
圖 十三、利用proteasome抑制劑(Lactacystin)處理人類嗜中性球後，G6PD缺乏的嗜中性球其Bcl-2不會因proteasome抑制劑(Lactacystin)的刺激而有回復的現象 49
圖 十四、利用proteasome抑制劑(MG132)在不同時間或不同濃度下處理人類嗜中性球後，G6PD缺乏的嗜中性球其Bcl-2不會因proteasome抑制劑(MG132)的處理而有回復的現象 50

1. Mortazavi Y, Chopra R, Gordon-Smith EC, Rutherford TR. Clonal patterns of X-chromosome inactivation in female patients with aplastic anaemia studies using a novel reverse transcription polymerase chain reaction method. Eur J Haematol. 2000;64:385-395.
2. Herschel M, Ryan M, Gelbart T, Kaplan M. Hemolysis and hyperbilirubinemia in an African American neonate heterozygous for glucose-6-phosphate dehydrogenase deficiency. J Perinatol. 2002;22:577-579.
3. Ho HY, Cheng ML, Chiu DT. Glucose-6-phosphate dehydrogenase--from oxidative stress to cellular functions and degenerative diseases. Redox Rep. 2007;12:109-118.
4. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64-74.
5. Fialkow L, Wang Y, Downey GP. Reactive oxygen and nitrogen species as signaling molecules regulating neutrophil function. Free Radic Biol Med. 2007;42:153-164.
6. Gao LP, Cheng ML, Chou HJ, Yang YH, Ho HY, Chiu DT. Ineffective GSH regeneration enhances G6PD-knockdown Hep G2 cell sensitivity to diamide-induced oxidative damage. Free Radic Biol Med. 2009;47:529-535.
7. Xu Y, Zhang Z, Hu J, et al. Glucose-6-phosphate dehydrogenase-deficient mice have increased renal oxidative stress and increased albuminuria. FASEB J. 2010;24:609-616.
8. Nikolaidis MG, Jamurtas AZ, Paschalis V, et al. Exercise-induced oxidative stress in G6PD-deficient individuals. Med Sci Sports Exerc. 2006;38:1443-1450.
9. Heistad DD, Wakisaka Y, Miller J, Chu Y, Pena-Silva R. Novel aspects of oxidative stress in cardiovascular diseases. Circ J. 2009;73:201-207.
10. Cheng ML, Ho HY, Wu YH, Chiu DT. Glucose-6-phosphate dehydrogenase-deficient cells show an increased propensity for oxidant-induced senescence. Free Radic Biol Med. 2004;36:580-591.
11. Wu YH, Cheng ML, Ho HY, Chiu DT, Wang TC. Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts. J Biomed Sci. 2009;16:18-25.
12. Ho HY, Cheng ML, Weng SF, et al. Glucose-6-phosphate dehydrogenase deficiency enhances enterovirus 71 infection. J Gen Virol. 2008;89:2080-2089.
13. Wu YH, Tseng CP, Cheng ML, Ho HY, Shih SR, Chiu DT. Glucose-6-phosphate dehydrogenase deficiency enhances human coronavirus 229E infection. J Infect Dis. 2008;197:812-816.
14. Ho HY, Cheng ML, Weng SF, Leu YL, Chiu DT. Antiviral effect of epigallocatechin gallate on enterovirus 71. J Agric Food Chem. 2009;57:6140-6147.
15. Pratico D. Evidence of oxidative stress in Alzheimer's disease brain and antioxidant therapy: lights and shadows. Ann N Y Acad Sci. 2008;1147:70-78.
16. Wan GH, Tsai SC, Chiu DT. Decreased blood activity of glucose-6-phosphate dehydrogenase associates with increased risk for diabetes mellitus. Endocrine. 2002;19:191-195.
17. Kleniewska P, Piechota A, Skibska B, Goraca A. The NADPH oxidase family and its inhibitors. Arch Immunol Ther Exp (Warsz). 2012;60:277-294.
18. Fan J, Frey RS, Malik AB. TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase. J Clin Invest. 2003;112:1234-1243.
19. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:245-313.
20. Tsai KJ, Hung IJ, Chow CK, Stern A, Chao SS, Chiu DT. Impaired production of nitric oxide, superoxide, and hydrogen peroxide in glucose 6-phosphate-dehydrogenase-deficient granulocytes. FEBS Lett. 1998;436:411-414.
21. Gupte RS, Floyd BC, Kozicky M, et al. Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver. Free Radic Biol Med. 2009;47:219-228.
22. Spolarics Z. Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid. J Leukoc Biol. 1998;63:534-541.
23. Flora SJ, Gautam P, Kushwaha P. Lead and ethanol co-exposure lead to blood oxidative stress and subsequent neuronal apoptosis in rats. Alcohol Alcohol. 2012;47:92-101.
24. Tuttle SW, Varnes ME, Mitchell JB, Biaglow JE. Sensitivity to chemical oxidants and radiation in CHO cell lines deficient in oxidative pentose cycle activity. Int J Radiat Oncol Biol Phys. 1992;22:671-675.
25. Ho HY, Wei TT, Cheng ML, Chiu DT. Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status. J Agric Food Chem. 2006;54:1638-1645.
26. Fico A, Paglialunga F, Cigliano L, et al. Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis. Cell Death Differ. 2004;11:823-831.
27. Mates JM, Segura JA, Alonso FJ, Marquez J. Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch Toxicol. 2008;82:273-299.
28. Shore GC, Nguyen M. Bcl-2 proteins and apoptosis: choose your partner. Cell. 2008;135:1004-1006.
29. Plati J, Bucur O, Khosravi-Far R. Apoptotic cell signaling in cancer progression and therapy. Integr Biol (Camb). 2011;3:279-296.
30. Jaeschke H, McGill MR, Ramachandran A. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity. Drug Metab Rev. 2012;44:88-106.
31. Matalova E, Svandova E, Tucker AS. Apoptotic signaling in mouse odontogenesis. OMICS. 2012;16:60-70.
32. Chen M, Wang J. Initiator caspases in apoptosis signaling pathways. Apoptosis. 2002;7:313-319.
33. Ploskonos MV. [The role of apoptosis Fas and Fasl markers in spermatogenesis]. Urologiia. 2012;1:77-80.
34. Bortner CD, Cidlowski JA. Life and death of lymphocytes: a volume regulation affair. Cell Physiol Biochem. 2011;28:1079-1088.
35. Estaquier J, Vallette F, Vayssiere JL, Mignotte B. The mitochondrial pathways of apoptosis. Adv Exp Med Biol. 2012;942:157-183.
36. Kouri FM, Jensen SA, Stegh AH. The role of Bcl-2 family proteins in therapy responses of malignant astrocytic gliomas: Bcl2L12 and beyond. ScientificWorldJournal. 2012;838:916-924.
37. Marzano AV, Frezzolini A, Caproni M, et al. Immunohistochemical expression of apoptotic markers in drug-induced erythema multiforme, Stevens-Johnson syndrome and toxic epidermal necrolysis. Int J Immunopathol Pharmacol. 2007;20:557-566.
38. Winfield LL, Payton-Stewart F. Celecoxib and Bcl-2: emerging possibilities for anticancer drug design. Future Med Chem. 2012;4:361-383.
39. Wang H, Takemoto C, Akasaka R, et al. Novel dimerization mode of the human Bcl-2 family protein Bak, a mitochondrial apoptosis regulator. J Struct Biol. 2009;166:32-37.
40. Bajwa N, Liao C, Nikolovska-Coleska Z. Inhibitors of the anti-apoptotic Bcl-2 proteins: a patent review. Expert Opin Ther Pat. 2012;22:37-55.
41. Milot E, Filep JG. Regulation of neutrophil survival/apoptosis by Mcl-1. ScientificWorldJournal. 2011;11:1948-1962.
42. Vogler M. BCL2A1: the underdog in the BCL2 family. Cell Death Differ. 2012;19:67-74.
43. Baliga BC, Kumar S. Role of Bcl-2 family of proteins in malignancy. Hematol Oncol. 2002;20:63-74.
44. Nys K, Agostinis P. Bcl-2 family members: essential players in skin cancer. Cancer Lett. 2012;320:1-13.
45. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645-654.
46. Martinou JC, Youle RJ. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cel. 2011;21:92-101.
47. Orwat DE, Batalis NI. Intravascular large B-cell lymphoma. Arch Pathol Lab Med. 2012;136:333-338.
48. Kelly PN, Strasser A. The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell Death Differ. 2011;18:1414-1424.
49. Breitschopf K, Haendeler J, Malchow P, Zeiher AM, Dimmeler S. Posttranslational modification of Bcl-2 facilitates its proteasome dependent degradation: molecular characterization of the involved signaling pathway. Mol Cell Biol. 2000;20:1886-1896.
50. Lutz RJ. Role of the BH3 (Bcl-2 homology 3) domain in the regulation of apoptosis and Bcl-2-related proteins. Biochem Soc Trans. 2000;28:51-56.
51. Glasgow JN, Qiu J, Rassin D, Grafe M, Wood T, Perez-Pol JR. Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain. Neurochem Res. 2001;26:647-659.
52. Jones GB, Mitchell MO, Weinberg JS, D'Amico AV, Bubley GJ. Towards enzyme activated antiprostatic agents. Bioorg Med Chem Lett. 2000;10:1987-1989.
53. Li B, Dou QP. Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci U S A. 2000;97:3850-3855.
54. Willimott S, Wagner SD. MiR-125b and miR-155 contribute to BCL2 repression and proliferation in response to CD40 ligand (CD154) in human leukemic B-cells. J Biol Chem. 2012;287:2608-2617
55. Badr G, Mohany M, Abu-Tarboush F. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-XL expression. Lipids Health Dis. 2011;10:236-244.
56. Haldar S, Jena N, Croce CM. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A. 1995;92:4507-4511.
57. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425-479.
58. Ebrahimi-Fakhari D, Wahlster L, McLean PJ. Protein degradation pathways in Parkinson's disease: curse or blessing. Acta Neuropathol. 2012 [Epub ahead of print]
59. Rahimi N. The ubiquitin-proteasome system meets angiogenesis. Mol Cancer Ther. 2012;11:538-548.
60. Busso CS, Wedgeworth CM, Izumi T. Ubiquitination of human AP-endonuclease 1 (APE1) enhanced by T233E substitution and by CDK5. Nucleic Acids Res. 2011;39:8017-8028.
61. Dennissen FJ, Kholod N, van Leeuwen FW. The ubiquitin proteasome system in neurodegenerative diseases: culprit, accomplice or victim? Prog Neurobiol. 2012;96:190-207.
62. Almond JB, Cohen GM. The proteasome: a novel target for cancer chemotherapy. Leukemia. 2002;16:433-443.
63. Ding F, Yin Z, Wang HR. Ubiquitination in Rho signaling. Curr Top Med Chem. 2011;11:2879-2887.
64. Piper RC, Lehner PJ. Endosomal transport via ubiquitination. Trends Cell Biol. 2011;21:647-655.
65. Susnow N, Zeng L, Margineantu D, Hockenbery DM. Bcl-2 family proteins as regulators of oxidative stress. Semin Cancer Biol. 2009;19:42-49.
66. Yang TM, Barbone D, Fennell DA, Broaddus VC. Bcl-2 family proteins contribute to apoptotic resistance in lung cancer multicellular spheroids. Am J Respir Cell Mol Biol. 2009;41:14-23.
67. Wang L, Azad N, Kongkaneramit L, et al. The Fas death signaling pathway connecting reactive oxygen species generation and FLICE inhibitory protein down-regulation. J Immunol. 2008;180:3072-3080.
68. Kongkaneramit L, Sarisuta N, Azad N, et al. Dependence of reactive oxygen species and FLICE inhibitory protein on lipofectamine-induced apoptosis in human lung epithelial cells. J Pharmacol Exp Ther. 2008;325:969-977.
69. Li D, Ueta E, Kimura T, Yamamoto T, Osaki T. Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination. Cancer Sci. 2004;95:644-650.
70. Azad N, Iyer AK, Manosroi A, Wang L, Rojanasakul Y. Superoxide-mediated proteasomal degradation of Bcl-2 determines cell susceptibility to Cr(VI)-induced apoptosis. Carcinogenesis. 2008;29:1538-1545.
71. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75:241-251.
72. Brinkmann V, Laube B, Abu Abed U, Goosmann C, Zychlinsky A. Neutrophil extracellular traps: how to generate and visualize them. J Vis Exp. 2010;36:36-39
73. Cheng ML, Ho HY, Huang YW, Lu FJ, Chiu DT. Humic acid induces oxidative DNA damage, growth retardation, and apoptosis in human primary fibroblasts. Exp Biol Med (Maywood). 2003;228:413-423.
74. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129-1132.
75. Clark M, Root RK. Glucose-6-phosphate dehydrogenase deficiency and infection: a study of hospitalized patients in Iran. Yale J Biol Med. 1979;52:169-179.
76. Chhabra R, Dubey R, Saini N. Gene expression profiling indicate role of ER stress in miR-23a~27a~24-2 cluster induced apoptosis in HEK293T cells. RNA Biol. 2011;8:648-664.