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系統識別號 U0026-0308201514362000
論文名稱(中文) 探討cAMP和ROS對於PMA誘導人類血癌細胞模型的細胞分化為巨核細胞之影響研究
論文名稱(英文) The effect of cAMP on the formation and functions of ROS during megakaryocyte differentiation of human erythroleukemia cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 103
學期 2
出版年 104
研究生(中文) 張嘉熙
研究生(英文) Ka-Hei Cheong
學號 S26025024
學位類別 碩士
語文別 中文
論文頁數 87頁
口試委員 指導教授-簡偉明
口試委員-錢偉鈞
口試委員-陳清玉
中文關鍵字 ROS  SOD2  cAMP  巨核細胞  分化 
英文關鍵字 ROS  SOD2  cAMP  megakaryocyte  differentiation 
學科別分類
中文摘要 血小板主要是經由巨核細胞的分化所生成,而巨核細胞的分化過程稱為Megakaryocytopoiesis。在不正常的巨核細胞分化過程會導致一些疾病的發生,如原發性血小板增多症(Essential thrombocythemia)與血小板低下症(Thrombocytopenia),這些類型的病患骨髓中會有異常增多或減少的巨核細胞或血小板。因此,研究巨核細胞的分化過程是有其必要性的。過去已知利用人類血癌細胞株 HEL和K562在PMA誘導下,會抑制細胞的增生而使細胞變大、增加染色體套數以及細胞表面抗原CD41/61的表達等巨核細胞分化時的特徵。且過去研究指出,ROS會參與在巨核細胞分化中,並且會增加表面抗原CD41/61的表達;而先前實驗室也發現cAMP會阻止巨核細胞分化。所以在本研究中,利用HEL細胞作為本研究的實驗模型來探討cAMP和ROS產生的之間的關係,並利用腺苷酸環化酶(Adenylyl cyclase)的活化劑forskolin(FSK)前處理在HEL細胞中來觀察cAMP是如何去影響ROS的體內平衡導致去影響一系列的巨核細胞分化過程。首先利用流式細胞儀分析發現經由PMA處理後ROS中的superoxide (O2-) 和hydrogen peroxide (H2O2)呈現time-dependently的增加。有趣地是我們也發現當前處理forskolin後雖然會增加H2O2的表現量但卻也會減少O2-的表現量。另一方面在西方墨點法分析中我們觀察到經由PMA誘導下SOD2則會呈現time-dependently的減少,而當處理forskolin後其SOD2的蛋白質表現量有被恢復回來,但SOD1的蛋白質表現量卻不受影響。進一步我們使用了MEK1/2的抑制劑結果顯示會降低forskolin恢復SOD2的蛋白質表現量的能力,最後再用SOD activity assay方法也能觀察到當經由PMA誘導下SOD2酶的活性被抑制而加入了forskolin後其活性有被恢復。因此,綜合上述所觀察到的結果,forskolin會直接通過活化MAPK的路徑來調控SOD2以及導致H2O2的增加。
英文摘要 Normal platelet production is largely affected by the megakaryocyte (MKs) differentiation. Abnormal differentiation process could result in clinical disorders like thrombocytopenia and essential thrombocythemia. Thus, the study of how megakaryocyte differentiation is essential. Phorbol 12-myristate 13-acetate (PMA) induced megakaryocytopoiesis in HEL or K562 cells as characterized by the inhibition of cell proliferation, increase of cell size, nuclear polyploidization and cell surface marker CD41/CD61. Previous studies have shown that ROS is involved in MKs differentiation, with an increase in CD41and CD61 expression. Therefore, in this study, the relation between cAMP and ROS production was investigated. Adenylyl cyclase activator forskolin(FSK) pretreated HEL and K562 cells were used as the model system to study how cAMP affects ROS homeostasis and subsequently affects MKs differentiation. Our data showed that superoxide and hydrogen peroxide were time-dependently induced by PMA treatment. Interestingly, we found that adenylyl cyclase activator forskolin not only increased PMA-induced hydrogen peroxide but also attenuated superoxide level in human erythroleukemia cells. On the other hand, we also observed that SOD2 expression level was decreased by PMA treatment, while SOD2 enzyme expression was restored in the presence of forskolin but did not affect SOD1 level. Furthermore, pretreatment the ERK1/2 inhibitor decreased the ability of forskolin in restoring SOD2 protein level. Thus, our data suggested that forskolin action was mediated through the activation of MAPK pathway to upregulate SOD2 protein level and led to increased hydrogen peroxide.
論文目次 口試合格證明............................................II
中文摘要...............................................III
Extended Abstract.......................................IV
誌謝...................................................XII
目錄..................................................XIII
圖目錄.................................................XIV
縮寫表.................................................XVI
緒論.....................................................1
材料與方法..............................................14
實驗結果................................................25
討論....................................................30
結論....................................................34
參考文獻................................................36
附錄....................................................47
參考文獻 [1] Pang L, Weiss MJ, and Poncz M. (2005) Megakaryocyte biology and related disorders. J Clin Invest. 115(12):3332-3338.
[2] Vögtle T, Cherpokova D, Bender M, Nieswandt B. (2015) Targeting platelet receptors in thrombotic and thrombo-inflammatory disorders. Hamostaseologie. 29;35(2).
[3] Leo D. Wang and Amy J. Wagers. (2011)Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nature Reviews Molecular Cell Biology.12, 643–655.
[4] Ravid K, Lu J, Zimmet JM, Jones MR. (2002)Roads to polyploidy: the megakaryocyte example. J Cell Physiol. 190(1):7-20.
[5] Teramura M, Kobayashi S, Hoshino S. (1992)Interleukin-11 enhances human megakaryocytopoiesis in vitro. Blood. 79:327.
[6] Chou FS, Mulloy JC. (2011)The thrombopoietin/MPL pathway in hematopoiesis and leukemogenesis. Journal of Cellular Biochemistry. 112(6):1491–1498.
[7] Kamonnaree Chotinantakul and Wilairat Leeanansaksiri. (2012)Hematopoietic StemCell Development, Niches, and Signaling Pathways. Bone Marrow Research. 270425.
[8] Royer Y, Staerk J, Costuleanu M, Courtoy PJ, Constantinescu SN. (2005)Janus kinases affect thrombopoietin receptor cell surface localization and stability. J Biol Chem. 280(29):27251-61.
[9] Kirito K, Osawa M, Morita H, Shimizu R, Yamamoto M, Oda A, Fujita H, Tanaka M, Nakajima K, Miura Y, Ozawa K, Komatsu N.(2002)A functional role of Stat3 in in vivo megakaryopoiesis. Blood. 99(9):3220-7.
[10] Snow JW, Abraham N, Ma MC, Abbey NW, Herndier B, Goldsmith MA. (2002)STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells. Blood. 99(1):95-101.
[11] Liu ZJ, Italiano J Jr, Ferrer-Marin F, Gutti R, Bailey M, Poterjoy B, Rimsza L, Sola-Visner M. (2011)Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated
GATA-1 levels in neonatal megakaryocytes. Blood. 117(15):4106-17.
[12] Nakao T, Geddis AE, Fox NE, Kaushansky K. (2008)PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27(Kip1). Cell Cycle. 7(2):257-66.
[13] Rojnuckarin P, Drachman JG, Kaushansky K. (1990) Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis. Blood. 94:1273–82.
[14] Garcia J, de Gunzburg J, Eychène A, Gisselbrecht S,Porteu F. (2001)Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf 1 and Rap1-B Raf-dependent pathways. Mol Cell Biol. 21:2659–70.
[15] Tamihiro Kamata, Catrin A. Pritchard and Andrew D. Leavitt. (2004) Raf-1 is not required for megakaryocytopoiesis or TPO-induced ERK phosphorylation. Blood. 103:2568-2570.
[16] Ezumi Y, Uchiyama T, Takayama H. (1998) Thrombopoietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets. Eur J Biochem. 258(3):976-85.
[17] Christopher M. Williams, Matthew T. Harper, and Alastair W. Poole. (2014)PKCα negatively regulates in vitro proplatelet formation and in vivo platelet production in mice. Platelets.Vol. 25, No. 1 , Pages 62-68.
[18] Klimchenko O, Mori M, DiStefano A, Langlois T. (2009)A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell–derived primitive hematopoiesis. Blood. 114(8):1506-1517.
[19] García P, Calés C. (1996)Endoreplication in megakaryoblastic cell lines is accompanied by sustained expression of G1/S cyclins and downregulation of cdc25C. Oncogene. 13(4):695-703.
[20] Long, M. W., Heffner, C. H., Williams, J. L., Peters, C. and Prochownik, E. V. (1990)Regulation of megakaryocyte phenotype in human erythroleukemia cells. J Clin Invest. 85(4), 1072-1084.
[21] Martin P, Papayannopoulou T. (1982)HEL Cells: A New Human Erythroleukemia Cell Line with Spontaneousand Induced Globin Expression. Science. 216(4551):1233-5.
[22] Tsuji T, Waga I, Tezuka K, Kamada M, Yatsunami K, Kodama H. (1998)Integrin ß2(CD18)-Mediated Cell Proliferation of HEL Cells on a Hematopoietic-Supportive Bone Marrow Stromal Cell Line, HESS-5 Cells. Blood. 91(4):1263-71.
[23] Chow DC, Wenning LA, Miller WM, Papoutsakis ET. (2001)Modeling pO(2) distributions in the bone marrow hematopoietic compartment.II. Modified Kroghian models. Biophys J. 81:685–696.
[24] Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD. (2007)Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc Natl Acad Sci USA. 104:5431–5436.
[25] Ivanovic Z, Dello Sbarba P, Trimoreau F, Faucher JL, Praloran V. (2000)Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion. 40:1482–1488.
[26] Norifumi Urao, MD, PhD and Masuko Ushio-Fukai, PhD.
(2013)Redox Regulation of Stem/Progenitor Cells and Bone Marrow Niche. Free Radic Biol Med. 54:26–39.
[27] S Chen, Y Su and J Wang. (2013)ROS-mediated platelet generation: a microenvironment-dependent manner formegakaryocyte proliferation, differentiation, and maturation. Cell Death and Disease. 4, e722.
[28] Yalcin S, Marinkovic D, Mungamuri SK, Zhang X, Tong W, Sellers R et al. (2010)ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3(-/-) mice. EMBO J. 29:4118–4131.
[29] Miyamoto K, Araki KY, Naka K, Arai F, Takubo K, Yamazaki S et al. (2007)Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell. 1:101–112.
[30] Jing Z, Ping Z, Yi L, Yin X, Jie W, Lingbo L. (2011)The cross-talk between ROS and p38MAPKα in the ex vivo expanded human umbilical cord blood CD133+ cells. J Huazhong Univ Sci Technol. 31:591–595.
[31] Abbas HA, Maccio DR, Coskun S, Jackson JG, Hazen AL, Sills TM et al. (2010)Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity. Cell Stem Cell. 7:606–617.
[32] Sardina JL, Lopez-Ruano G, Sanchez-Abarca LI, Perez-Simon JA, Gaztelumendi A, Trigueros C et al. (2010)p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation. Cell Death Differ. 17:1842–1854.
[33] Nakata S, Matsumura I, Tanaka H, Ezoe S, Satoh Y, Ishikawa J et al. (2004)NF-kB family proteins participate in multiple steps of hematopoiesis through elimination of reactive oxygen species. J Biol Chem. 279:55578–55586.
[34] Urao N, McKinney RD, Fukai T, Ushio-Fukai M. (2012)NADPH oxidase 2 regulates bone marrow microenvironment following hindlimb ischemia: role in reparative mobilization of progenitor cells. Stem Cells. 30:923–934.
[35] José L. Sardina, Guillermo López-Ruano, Beatriz Sánchez-Sánchez, Marcial Llanillo, Angel Hernández-Hernández. (2012) Reactive oxygen species: Are they important for haematopoiesis? Crit Rev Oncol Hematol. 81:257–274.
[36] Jiang F, Zhang Y, Dusting GJ. (2011)NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol Rev. 63:218–242.
[37] Lassègue B, SanMartín A, Griendling KK. (2012)Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ Res. 110:1364–1390.
[38] Diaz B, Courtneidge SA. (2012)Redox signaling at invasive microdomains in cancer cells. Free Radic Biol Med. 52:247–256.
[39] Sareila O, Kelkka T, Pizzolla A, Hultqvist M, Holmdahl R. (2011)NOX2 complex-derived ROS as immune regulators. Antioxid Redox Signal. 5:2197–2208.
[40] Carl Nathan, Aihao Ding. (2010)SnapShot: Reactive Oxygen Intermediates (ROI). Cell. 140:951–951.e952.
[41] Fukai T, Ushio-Fukai M. (2011)Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal. 15:1583–1606.
[42] Pervaiz S, Taneja R, Ghaffari S. (2009)Oxidative stress regulation of stem and progenitor cells. Antioxid Redox Signal. 11:2777–2789.
[43] Carine Michiels, Michiels Raes, Olivier Toussaint, José Remacle. (1994)Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med. 17:235–248.
[44] Elisabeth Dernbach, Carmen Urbich, Ralf P. Brandes, Wolf K. Hofmann, Andreas M. Zeiher, and Stefanie Dimmeler. (2004)Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood. 104:3591–3597.
[45] Hayes JD, McMahon M. (2009)NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci. 34(4):176–188.
[46] Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS. (2009)Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature. 461(7260):109–113.
[47] Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, Yamamoto M, Motohashi H. (2012)Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell. 22(1):66–79.
[48] DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, Mangal D, Yu KH, Yeo CJ, Calhoun ES, Scrimieri F, Winter JM, Hruban RH, Iacobuzio-Donahue C, Kern SE, Blair IA, Tuveson DA. (2011)Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature. 475(7354):106–109.
[49] Ren D, Villeneuve NF, Jiang T, Wu T, Lau A, Toppin HA, Zhang DD. (2011)Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc Natl Acad Sci USA. 108(4):1433–1438.
[50] Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, Tolliday NJ, Golub TR, Carr SA, Shamji AF, Stern AM, Mandinova A, Schreiber SL, Lee SW. (2011)Selective killing of cancer cells by a small molecule targeting the stress
response to ROS. Nature. 475(7355):231–234.
[51] Somwar R, Erdjument-Bromage H, Larsson E, Shum D, Lockwood WW, Yang G, Sander C, Ouerfelli O, Tempst PJ, Djaballah H, Varmus HE. (2011)Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines. Proc Natl Acad Sci USA. 108(39):16375–16380.
[52] Andrea Glasauer, Laura A. Sena, Lauren P. Diebold, Andrew P. Mazar, and Navdeep S. Chandel. (2014)Targeting SOD1 reduces experimental non–small-cell lung cancer. J Clin Invest.124(1):117–128.
[53] Hilton JB, White AR, Crouch PJ. (2015)Metal-deficient SOD1 in amyotrophic lateral sclerosis. J Mol Med (Berl). 93(5):481-7.
[54] Jang YC and Remmen VH. (2009)The mitochondrial theory of aging: insight from transgenic and knockout mouse models. Exp Gerontol. 44:256–260.
[55] Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH, Wallace DC, and Epstein CJ. (1995)Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 11:376–381.
[56] Sumitra Miriyala, Ivan Spasojevic, Artak Tovmasyan, Daniela Salvemini, Zeljko Vujaskovic, Daret St. Clair, and Ines Batinic-Haberle. (2012)Manganese superoxide dismutase, MnSOD and its mimics. Biochim Biophys Acta. 1822, 794-814.
[57] Nadine Hempel, Pauline M. Carrico, and J. Andres Melendez. A. (2011)Manganese superoxide dismutase (Sod2) and redox-control of signaling events that drive metastasis. Anticancer Agents Med Chem. 11, 191-201.
[58] Dongyun Zhang, Yulei Wang, Yuguang Liang, Min Zhang, Jinlong Wei, Xiao Zheng1, Fei Li, Yan Meng, Nina Wu Zhu, Jingxia Li, Xue-Ru Wu and Chuanshu Huang. (2014)Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration. Journal of Cell Science. 127, 2920–2933.
[59] Fukai T, Galis ZS, Meng XP, Parthasarathy S, and Harrison DG. (1998)Vascular expression of extracellular superoxide dismutase in atherosclerosis. J Clin Invest. 101:2101–2111.
[60] Strålin P, Karlsson K, Johansson BO, and Marklund SL. (1995)The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol. 15:2032–2036.
[61] Tan RJ, Lee JS, Manni ML, Fattman CL, Tobolewski JM, Zheng M, Kolls JK, Martin TR, and Oury TD. (2006)Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury. Am J Respir Cell Mol Biol. 34:226–232.
[62] Yuqi Cui, Fengpeng Jia, Jianfeng He, Xiaoyun Xie, Zhihong Li, Minghuan Fu, Hong Hao, Ying Liu, Dylan Z. Liu, Peter J. Cowan, Hua Zhu, Qinghua Sun, Zhenguo Liu. (2015) Ambient Fine Particulate Matter Suppresses In Vivo Proliferation of Bone Marrow Stem Cells through Reactive Oxygen Species Formation. PLoS ONE. 10(6):e0127309.
[63] Schwarz, U.R., Walter, U. and Eigenthaler, M. (2001) Taming platelets with cyclic nucleotides. Biochemical Pharmacology. 62, 1153–1161.
[64] Antonija Jurak Begonja, Stepan Gambaryan, Harald Schulze, Sunita Patel-Hett, Joseph E. Italiano Jr, John H. Hartwig, and Ulrich Walter. (2013)Differential roles of cAMP and cGMP in megakaryocyte maturation and platelet biogenesis. Exp Hematol. 41(1):91–101
[65] Zaher Raslan and Khalid M. Naseem. (2014)The control of blood platelets by cAMP signaling. Biochem Soc Trans. 42, 289–294.
[66] Ravid K, Lu J, Zimmet JM, Jones MR. (2002)Roads to polyploidy: the megakaryocyte example. J Cell Physiol. 190(1):7-20.
[67] Chang Y, Bluteau D, Debili N, Vainchenker W. (2007)From hematopoietic stem cells to platelets. J Thromb Haemost. 5 (Suppl. 1):318–27.
[68] Séverin S, Ghevaert C, Mazharian A. (2010)The mitogen-activated protein kinase signaling pathways: role in megakaryocyte differentiation. J Thromb Haemost. 8:17–26.
[69] Rajarshi Sengupta, Tao Sun, Nicole M Warrington, and Joshua B. Rubin. (2011)Treating brain tumors with PDE4 Inhibitors. Trends Pharmacol Sci. 32(6):337–344.
[70] Katsumi HIROSE, Satoru MONZEN, Haruka SATO, Mariko SATO, Masahiko AOKI, Yoshiomi HATAYAMA, Hideo KAWAGUCHI, Yuichiro NARITA, Yoshihiro TAKAI, and Ikuo KASHIWAKURA.
(2013)Megakaryocytic differentiation in human chronic myelogenous leukemia K562 cells induced by ionizing radiation in combination with phorbol 12-myristate 13-acetate. Journal of Radiation Research. 54, 438–446.
[71] Amaro-Ortiz A, Yan B, D'Orazio JA. (2014)Ultraviolet radiation, aging and the skin: prevention of damage by topical cAMP manipulation. Molecules. 19(5):6202-19.
[72] Fei Li, Tunan Chen, Shengli Hu, Jiangkai Lin, Rong Hu, Hua Feng. (2013)Superoxide Mediates Direct Current Electric Field-Induced Directional Migration of Glioma Cells through the Activation of AKT and ERK. PLoS ONE. 8(4):e61195.
[73] Mishima K, Baba A, Matsuo M, Itoh Y, Oishi R. (2006)Protective effect of cyclic AMP against cisplatin- induced nephrotoxicity. Free radical biology & medicine. 40(9):1564–1577.
[74] Tamura I, Sato S, Okada M, Tanabe M, Lee L, Maekawa R, Asada H, Yamagata Y, Tamura H, Sugino N. (2014)Importance of C/EBPβ binding and histone acetylation status in the promoter regions for induction of IGFBP-1, PRL, and Mn-SOD by cAMP in human endometrial stromal cells. Endocrinology. 155(1):275-86.
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