臺灣博碩士論文加值系統

細胞分化的過程是受到細胞內特定的基因活化互相合作所調控。根據實驗室之前的結果，我們發現到炭疽桿菌（Bacillus anthracis）分泌的致死毒素（lethal toxin，簡稱LT）會阻擋TPA （12-O-tetrdecanoylphorbol-13 acetate）所誘導的HEL（human erythroleukemia）細胞株往巨核細胞分化。經由分析微陣列（microarray）的數據，我們發現BHLHB2、DACH1、LIMK1、RBP5、RIS1等基因在TPA的處理下表現量增加，且在LT前處理後再處理TPA下表現量下降。所以我們假設這些基因可能在正常的巨核細胞分化及LT抑制的巨核細胞分化中扮演重要的角色。我們使用shRNA去分別抑制這些基因的表現，利用流式細胞儀分析TPA所誘發的分化情形是否有被阻擋的現象，我們發現BHLHB2、DACH1、RBP5等基因表現分別受抑制的時候，TPA所誘導的分化情形受到阻礙。根據實驗結果，我們得到的結論是BHLHB2、DACH1、RBP5等基因在巨核細胞的分化上扮演重要的角色。

The process of cell differentiation is regulated by the coordination of cell type-specific gene activation. Our previous studies indicated that anthrax lethal toxin (LT) could suppress TPA (12-O-tetradecanoylphorbol-13 acetate) induced megakaryocytic differentiation in human erythroleukemia (HEL) cell line. Based on microarray data, BHLHB2, DACH1, LIMK1, RBP5, RIS1 genes were up-regulated after TPA treatments and down-regulated upon LT-pretreatments, so we hypothesized that those genes may play important roles in normal and LT-suppressed megakaryocytic differentiation. Using flow cytometry, we found the differentiation abilities (specific surface marker expression and DNA polyploidy) of HEL cell were blocked when each of those genes was knockdown by shRNA. In this study, we have identified BHLHB2, DACH1, RBP5 genes play potential roles in megakaryocytic differentiation.

致謝 ----------------------------------------------------------------Ⅰ
Abstract ------------------------------------------------------------Ⅲ
中文摘要 ------------------------------------------------------------Ⅳ
Content -------------------------------------------------------------Ⅴ
Content of Figures and Tables ---------------------------------------Ⅵ
Introduction ---------------------------------------------------------1
Materials and Methods ------------------------------------------------9
Results -------------------------------------------------------------17
Discussion ----------------------------------------------------------28
References ----------------------------------------------------------31
Figures and Tables --------------------------------------------------36
Appendices ----------------------------------------------------------52

1. Deutsch VR, Tomer A. Megakaryocyte development and platelet production. Br J Haematol. 2006;134:453-466.
2. Hoffman R, Murrav LJ, Young JC, Luens KM, Bruno E. Hierarchical structure of human megakaryocyte progenitor cells. Stem Cells. 1996;14 Suppl 1:75-81.
3. Matsumura I, Kanakura Y. Molecular control of megakaryopoiesis and thrombopoiesis. Int J Hematol. 2002;75:473-483.
4. Nutt SL, Metcalf D, D'Amico A, Polli M, Wu L. Dynamic regulation of PU.1 expression in multipotent hematopoietic progenitors. J Exp Med. 2005;201:221-231.
5. Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and related disorders. J Clin Invest. 2005;115:3332-3338.
6. Miyazaki H. Physiologic role of TPO in thrombopoiesis. Stem Cells. 1996;14 Suppl 1:133-138.
7. Briddell RA, Brandt JE, Straneva JE, Srour EF, Hoffman R. Characterization of the human burst-forming unit-megakaryocyte. Blood. 1989;74:145-151.
8. Kaushansky K. Thrombopoietin. N Engl J Med. 1998;339:746-754.
9. Kaushansky K. The molecular mechanisms that control thrombopoiesis. J Clin Invest. 2005;115:3339-3347.
10. Kaushansky K. Historical review: megakaryopoiesis and thrombopoiesis. Blood. 2008;111:981-986.
11. Kaushansky K, Drachman JG. The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production. Oncogene. 2002;21:3359-3367.
12. Kirito K, Kaushansky K. Transcriptional regulation of megakaryopoiesis: thrombopoietin signaling and nuclear factors. Curr Opin Hematol. 2006;13:151-156.
13. Chiang C-Y. Effects of Anthrax Lethal Toxin on Megakaryocytopoiesis - A Proteomic Approach. Department of Human genetics Hualien : Tzu Chi University. 2006.
14. Tsai M-F. The Effects of Bacillus anthracis Lethal Toxin on Megakaryocyte Differentiation. Department of Human genetics Hualien : Tzu Chi University. 2004.
15. Chung C-P. The Effects of Bacillus anthracis Lethal Toxin on Megakaryocytic Polyploidization. Department of Human genetics Hualien : Tzu Chi University. 2004.
16. Tournier JN, Quesnel-Hellmann A, Cleret A, Vidal DR. Contribution of toxins to the pathogenesis of inhalational anthrax. Cell Microbiol. 2007;9:555-565.
17. Banks DJ, Ward SC, Bradley KA. New insights into the functions of anthrax toxin. Expert Rev Mol Med. 2006;8:1-18.
18. Abrami L, Reig N, van der Goot FG. Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol. 2005;13:72-78.
19. Young JA, Collier RJ. Anthrax toxin: receptor binding, internalization, pore formation, and translocation. Annu Rev Biochem. 2007;76:243-265.
20. Duesbery NS, Webb CP, Leppla SH, et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science. 1998;280:734-737.
21. Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, Montecucco C. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun. 1998;248:706-711.
22. Nishida E, Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993;18:128-131.
23. Neiman AM. Conservation and reiteration of a kinase cascade. Trends Genet. 1993;9:390-394.
24. Melemed AS, Ryder JW, Vik TA. Activation of the mitogen-activated protein kinase pathway is involved in and sufficient for megakaryocytic differentiation of CMK cells. Blood. 1997;90:3462-3470.
25. Whalen AM, Galasinski SC, Shapiro PS, Nahreini TS, Ahn NG. Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase. Mol Cell Biol. 1997;17:1947-1958.
26. Dixon TC, Meselson M, Guillemin J, Hanna PC. Anthrax. N Engl J Med. 1999;341:815-826.
27. Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A. 1982;79:3162-3166.
28. Leppla SH. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:189-198.
29. Friedlander AM. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem. 1986;261:7123-7126.
30. O'Brien J, Friedlander A, Dreier T, Ezzell J, Leppla S. Effects of anthrax toxin components on human neutrophils. Infect Immun. 1985;47:306-310.
31. Abi-Habib RJ, Urieto JO, Liu S, Leppla SH, Duesbery NS, Frankel AE. BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin. Mol Cancer Ther. 2005;4:1303-1310.
32. Alileche A, Serfass ER, Muehlbauer SM, Porcelli SA, Brojatsch J. Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response. PLoS Pathog. 2005;1:e19.
33. Kirby JE. Anthrax lethal toxin induces human endothelial cell apoptosis. Infect Immun. 2004;72:430-439.
34. Pandey J, Warburton D. Knock-on effect of anthrax lethal toxin on macrophages potentiates cytotoxicity to endothelial cells. Microbes Infect. 2004;6:835-843.
35. Wu AG, Alibek D, Li YL, Bradburne C, Bailey CL, Alibek K. Anthrax toxin induces hemolysis: an indirect effect through polymorphonuclear cells. J Infect Dis. 2003;188:1138-1141.
36. During RL, Li W, Hao B, et al. Anthrax lethal toxin paralyzes neutrophil actin-based motility. J Infect Dis. 2005;192:837-845.
37. Kassam A, Der SD, Mogridge J. Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin. Cell Microbiol. 2005;7:281-292.
38. Paccani SR, Tonello F, Ghittoni R, et al. Anthrax toxins suppress T lymphocyte activation by disrupting antigen receptor signaling. J Exp Med. 2005;201:325-331.
39. Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci U S A. 1993;90:2291-2294.
40. Kau JH, Sun DS, Tsai WJ, et al. Antiplatelet activities of anthrax lethal toxin are associated with suppressed p42/44 and p38 mitogen-activated protein kinase pathways in the platelets. J Infect Dis. 2005;192:1465-1474.
41. Kau JH, Lin CG, Huang HH, et al. Calyculin A sensitive protein phosphatase is required for Bacillus anthracis lethal toxin induced cytotoxicity. Curr Microbiol. 2002;44:106-111.
42. Mikesova E, Barankova L, Sakmaryova I, Tatarkova I, Seeman P. Quantitative multiplex real-time PCR for detection of PLP1 gene duplications in Pelizaeus-Merzbacher patients. Genet Test. 2006;10:215-220.
43. Park JM, Greten FR, Li ZW, Karin M. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science. 2002;297:2048-2051.
44. Long MW, Heffner CH, Williams JL, Peters C, Prochownik EV. Regulation of megakaryocyte phenotype in human erythroleukemia cells. J Clin Invest. 1990;85:1072-1084.
45. Papayannopoulou T, Nakamoto B, Yokochi T, Chait A, Kannagi R. Human erythroleukemia cell line (HEL) undergoes a drastic macrophage-like shift with TPA. Blood. 1983;62:832-845.
46. Hogge D, Fanning S, Bockhold K, et al. Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol. 1997;96:790-800.
47. Bruno E, Briddell R, Hoffman R. Effect of recombinant and purified hematopoietic growth factors on human megakaryocyte colony formation. Exp Hematol. 1988;16:371-377.
48. Kaushansky K, Broudy VC, Lin N, et al. Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development. Proc Natl Acad Sci U S A. 1995;92:3234-3238.
49. Teramura M, Kobayashi S, Hoshino S, Oshimi K, Mizoguchi H. Interleukin-11 enhances human megakaryocytopoiesis in vitro. Blood. 1992;79:327-331.
50. Lai Y-L. Association of MAPK Pathway on anthracis Lethal Toxin Suppressed Megakaryocytic Differentiation. Department of Human genetics Hualien : Tzu Chi University. 2007.
51. Iwata T, Kawamoto T, Sasabe E, et al. Effects of overexpression of basic helix-loop-helix transcription factor Dec1 on osteogenic and adipogenic differentiation of mesenchymal stem cells. Eur J Cell Biol. 2006;85:423-431.
52. Shen M, Kawamoto T, Yan W, et al. Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes. Biochem Biophys Res Commun. 1997;236:294-298.
53. Mardon G, Solomon NM, Rubin GM. dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila. Development. 1994;120:3473-3486.
54. Zhang L, E X, Luker KE, et al. Analysis of human cellular retinol-binding protein II promoter during enterocyte differentiation. Am J Physiol Gastrointest Liver Physiol. 2002;282:G1079-1087.
55. Sun H, Lu B, Li RQ, Flavell RA, Taneja R. Defective T cell activation and autoimmune disorder in Stra13-deficient mice. Nat Immunol. 2001;2:1040-1047.
56. Cavalloni G, Dane A, Piacibello W, et al. The involvement of human-nuc gene in polyploidization of K562 cell line. Exp Hematol. 2000;28:1432-1440.
57. Guerriero R, Mattia G, Testa U, et al. Stromal cell-derived factor 1alpha increases polyploidization of megakaryocytes generated by human hematopoietic
progenitor cells. Blood. 2001;97:2587-2595.
58. Jung YJ, Chae HC, Seoh JY, et al. Pim-1 induced polyploidy but did not affect megakaryocytic differentiation of K562 cells and CD34+ cells from cord blood. Eur J Haematol. 2007;78:131-138.
59. Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci U S A. 1980;77:2936-2940.
60. Zhou GB, Zhang J, Wang ZY, Chen SJ, Chen Z. Treatment of acute promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide: a paradigm of synergistic molecular targeting therapy. Philos Trans R Soc Lond B Biol Sci. 2007;362:959-971.