臺灣博碩士論文加值系統

全反式維甲酸已被證實可以成功治療急性前骨髓性白血病，然這樣的治療卻可能導致高達百分之三十的病患伴隨有稱為分化症候群的併發症，大量經全反式維甲酸誘導分化的前骨髓白血病細胞灌注至肺泡腔中而導致臨床上類似急性肺發炎的症狀。這些浸潤至肺泡腔中的細胞如何被清除目前尚未完全明瞭，過去研究指出凋亡的已分化前骨髓性細胞與肺泡中巨噬細胞的相互作用對於分化症候群恢復期扮演重要角色。

本研究中，我們將進一步探討凋亡的全反式維甲酸治療之急性前骨髓性白血球細胞(ATRA-NB4)與肺泡巨噬細胞(NR8383)作用的機制。首先使用流式細胞利用Annexin V與7-AAD觀察細胞死亡狀況。確認細胞凋亡後接著收集凋亡細胞的細胞條件液(condition medium, CM)，觀察這些細胞條件液對於細胞貼附與遷移活性的影響。根據結果顯示，NR8383細胞可以快速將凋亡ATRA-NB4吞噬。此外，凋亡細胞的細胞條件液也被證實可藉由抗遷移以及抑制ATRA-NB4貼附至人類臍靜脈內皮細胞(human umbilical vein endothelial cells , HUVEC)的活性達到抗發炎的效果。

下一部分實驗，我們接著探討在其中扮演抗發炎作用以及促進巨噬細胞吞噬的調節物。過去已知Annexin A1在糖皮質固醇所誘導的抗發炎作用扮演重要角色。實驗結果顯示，凋亡ATRA-NB4細胞可以藉由microparticle的方式將Annexin A1釋放，此外也會減少促發炎細胞激素IL-8的釋放量。

根據以上結果可知，凋亡ATRA-NB4細胞可以藉由釋放Annexin A1(+) microparticle與抑制IL-8釋出達到抗發炎效果。在分化症候群的恢復期，可以促進細胞清除且避免細胞再度浸潤至肺泡腔中。因此，這樣的發現未來也可能可以應用在分化症候群的治療上，促進分化症候群所導致急性肺發炎的恢復，避免分化症候群嚴重度。

All- trans retinoic acid (ATRA) has been used successfully in the treatment of acute promyelocytic leukemia (APL). However, this treatment may complicate with differentiation syndrome (DS) in 30% of patients, which manifests with clinical picture of acute lung injury and massive infiltration of ATRA-treated APL (ATRA-APL) cells into alveolar spaces. It is not clear how these ATRA-APL cells were cleared during resolution phase of DS. Previous studies have reported that cross-talk between apoptotic ATRA-APL cells and alveolar macrophage is crucial during the resolution phase of DS.

In this study, we investigated the mechanism underlying the interaction between apoptotic ATRA-APL (NB4) cells and alveolar macrophages (NR8383). Different stages of apoptotic cells were determined by flowcytometry by using antibodies specific to annexin V and 7-AAD. Condition medium (CM) derived from apoptotic ATRA-NB4 cells was harvested to determine its biological activity by transmigration assay and adhesion assay. Our results indicated that NR8383 cells were able to engulf apoptotic ATRA-NB4 cells.

Furthermore, the CM derived from apoptotic ATRA-NB4 cells had anti-inflammatory activity as evidenced by inhibiting the recipient cells migration and adhesion to human umbilical vein endothelial cells (HUVEC). Further studies we focused on investigating the mediators responsible for the anti-inflammatory activities in the CM which facilitate the phagocytosis activity of alveolar macrophage. Annexin A1 is a mediator that regulates the immune response of glucocorticoids. We found that apoptotic ATRA-NB4 cells would release Annexin A1 via microparticle (MP). On the other hand, the release of the pro-inflammatory cytokine: IL-8 will diminish as well.

Based on these finding, we conclude that the CM collected from apoptotic ATRA-NB4 cells has anti-inflammatory activities by releasing AnxA1(+) MP and reducing the release of IL-8. During the resolution stage of DS, these apoptotic cells could increase clearance of dying cells and prevent further infiltration in alveolar. Thus, these findings would suggest the potential therapeutic strategies to modulate the resolution of acute lung injury in DS.

致謝 4
中文摘要 5
英文摘要 7
壹、文獻背景 9
貳、研究重要性與目的 25
参、實驗材料與方法 29
一、細胞之準備與培養 30
二、細胞數的調整 31
三、藥物配製 32
四、流式細胞儀分析(flow cytometry assay) 32
五、凋亡細胞(apoptotic cell)流式細胞儀分析 33
六、吞噬行為測定(phagocytosis assay) 33
七、細胞遷移之測量 (transmigration assay)及嵌入培養結果之讀取 34
八、細胞貼附能力分析(cell adhesion assay) 34
九、Microparticles的萃取與分析 35
十、Annexin A1含量測定 35
十一、細胞激素含量測定 36
十二、資料分析與統計 36
肆、實驗結果 37
一、紫外線照射後誘導全反式維甲酸治療的NB4細胞走向凋亡(UV induced ATRA-NB4 cell apoptosis) 38
二、紫外線誘發細胞凋亡的細胞條件液具有抗貼附以及抗遷移活性(The condition medium collected from UV-induced apoptotic ATRA-NB4 had the anti-adhesion and anti-migration activity) 39
三、NB4細胞以及經全反式維甲酸治療後的NB4細胞經紫外線照射後細胞條件液中Annexin A1的濃度下降(The concentration of Annexin A1 released from UV-induced apoptotic NB4 and ATRA-NB4 cell decreased ) 42
四、凋亡細胞釋出microparticle表面帶有Annexin A1的比例增加並且釋出IL-8 的量減少(The released percentage of Annexin A1(+) MP increased but free from IL-8 decreased from apoptotic cells) 43
伍、實驗圖表與說明 45
陸、討論 58
柒、參考文獻 64

1. LK, H., Acute promyelocytic leukemia. Acta Med Scand, 1957. 159: p. 189-194.

2. Wang ZY, C.Z., Acute promyelocytic leukemia: from highly fatal to highly curable, Blood. 2008. p. 2505-2515.

3. Warrel RP, J.d.T.H., Wang ZY, Degos L, Acute promyelocytic leukemia. N Engl J Med, 1993. 329: p. 177-189.

4. de The H, L.C., Marchio A, The PML-RARa fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell, 1991. 66: p. 675-684.

5. de The H, C.C., Lanotte M, Degos L, Dejean A, The t(15;17) translocation of acute promyelocytic leukemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus. Nature, 1990. 347: p. 558-561.

6. de The H, c.Z., Acute promyelocytic leukemia : novel insights into the mechanisms of cure. Nat Rev Cancer, 2010. 10: p. 775-783.

7. Huang ME, Y.Y., Chen SR,Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY, Use of all-trans retinoic acid in treatment of acute promyelocytic leukemia. Blood, 1988. 72: p. 567-572.

8. Tallman MS, N.C., Feusner JH, Rowe JM, Acute promyelocytic leukemia: evolving therapeutic strategies. Blood, 2002. 99: p. 759-767.

9. Nasr R, L.-B.V., Zhu J, Guillemin MC, de The H, Therapy-induced PML/RARA proteolysis and acute promyelocytic leukemia cure. Clin Cancer Res, 2009. 15: p. 6321-6326.

10. Wang ZY, C.Z., Acute promyelocytic leujemia: from highly fatal to highly curable. Blood, 2008. 111: p. 2505-2515.

11. Pei-Zheng Zheng, K.-K.W., Qun-Ye Zhang, Qiu-Hua Huang, Yan-Zhi Du, Qing-Hua Zhang, Da-Kai Xiao, Shu-Hong Shen, Sandrine Imbeaud, Eric Eveno, Chun-Jun Zhao, Yu-Long Chen, Hui-Yong Fan, Samuel Waxman, Charles Auffray, Gang Jin, Sai-Juan Chen, Zhu Chen, Ji Zhang, Systems analysis of transcriptome and proteome in retinoic acid arsenic trioxide-induced cell differentiation apoptosis of promyelocytic leukemia. PNAS, 2005. 102: p. 7653-7658.

12. Degos L, W.Z., All-trans retinoic acid in acute promyelocytic leukemia. Oncogene, 2001. 20: p. 7140-7145.

13. Frankel SR, E.A., Lauwers G, Weiss M, Warrell RP, The "retinoic acid syndrome" in acute promyelocytic leukemia. Ann Intern Med, 1992. 117: p. 292-296.

14. Tallman MS, A.J., Schiffer CA, Appelbaum FR, Feusner JH, Ogden A, Clinical description of 44 patients with acute promyelocytic leukemia who developed the retinoic acid syndrome. Blood, 2000. 1: p. 90-95.

15. Eduardo Magalhães Rego, G.C.D.S., Differentiation Syndrome in Promyelocytic Leukemia: Clinical Presentation, Pathogenesis and Treatment. Mediterr J Hematol Infect Dis, 2011. 3: p. Open Journal System.

16. Viola A, L.A., Chemokines and thir receptors: drug targets in immunity and inflammation. Annu Rev Pharmacol Toxicol, 2008. 48: p. 171-197.

17. Tsai WH, H.H., Lin CC, Ho CK, Kou YR., Role of interleukin-8 and growth-regulated oncogene-alpha in the chemotactic migration of all-trans retinoic acid-treated promyelocytic leukemic cells toward alveolar epithelial cells. Crit Care Med. , 2007. 35: p. 879-885.

18. Puneet P, M.S., Bhatia M, Chemokines in acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol, 2005. 288: p. 3-15.

19. Luesink M, P.J., Wissink WM, Linssen PC, Muus P, Pfundt R, de Witte TJ, van der Reijden BA, Jansen JH., Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia: triggering the differentiation syndrome. Blood, 2009. 114: p. 5512-5521.
20. Fenaux P, C.C., Degos L., All-trans retinoic acid and chemotherapy in the treatment of acute promyelocytic leukemia. Hematol, 2001. 38: p. 13-25.

21. Schuster, M.H.K.a.D.P., The acute respiratory distress syndrome. N Engl J Med, 1995. 332: p. 27-37.

22. Reutershan, F.M.K.a.J., CXCR2 in Acute Lung Injury. Mediators of Inflammation, 2012. 2012: p. 1-8.

23. G. D. Rubenfeld, E.C., E. Peabody, Incidence and outcomes of acute lung injury. N Engl J Med, 2005. 353: p. 1685-1693.

24. Matthay, M.C.a.M.A., Pharmacotherapy of acute lung injury and the acute respiratory distress syndrome. J Intensive Care Med, 2006. 21: p. 119-143.

25. L. J. Quinton, S.N., P. Zhang, K. I. Happel, L. Gamble, and G. J. Bagby, Effects of systemic and local CXC chemokine administration on the ethanol-induced suppression of pulmonary neutrophil recruitment. Alcoholism, 2005. 29: p. 1198-1205.

26. Abraham, E., Neutrophils and acute lung injury. Crit Care Med, 2003. 31: p. 195-199.

27. H. E. Broxmeyer, B.S.Y., C. Kim, G. Hangoc, S. Cooper, and C. Mantel,, Chemokine regulation of hematopoiesis and the involvement of pertussis toxin-sensitive Gαi proteins. Annals of the New York Academy of Sciences, 2001. 938: p. 117-128.

28. Butcher, J.J.C .E.C., Chemokines in tissuespecific and microenvironment-specific lymphocyte homing. Immunology, 2000. 12: p. 336-341.

29. C. H. Kim, E.C.B., and B. Johnston, Distinct subsets of human Vα24-invariant NKT cells: cytokine responses and chemokine receptor expression. Immunology, 2002. 23: p. 516-519.

30. Kerr, J.F., Wyllie, A. H., and Currie, A. R, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972. 26: p. 239-257.

31. Norbury, C.J., and Hickson, Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol, 2001. 41: p. 367-401.

32. ELMORE, S., Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol, 2007. 35: p. 495-516.

33. Vermes, I., Haanen, C., and Reutelingsperger, C., Flow cytometry of apoptotic cell death. . J. Immunol. Methods, 2000. 243: p. 167-190.

34. Savill, J., and Fadok, Corpse clearance defines the meaning of cell death. Nature, 2000. 407: p. 784-788.

35. Kurosaka, K., Takahashi, M., Watanabe, N., and Kobayashi, Y, Silent cleanup of very early apoptotic cells by macrophages. J Immunol, 2003. 171: p. 4672-4679.

36. Igney, F.H., and Krammer, P. H., Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer, 2002. 2: p. 277-288.

37. Martinvalet, D., Zhu, P., and Lieberman, J, GranzymeAinduces caspaseindependent mitochondrial damage, a required first step for apoptosis. Immunity 2005. 22: p. 355-370.

38. Whyte MKB, M.L., MacDermot J and Haslett C Impairment of function in aging neutrophils is associated with apoptosis. J. Immunol, 1993. 150: p. 5124-5134.

39. Takizawa F, T.S.a.N.S., Enhancement of macrophage phagocytosis upon iC3b deposition on apoptotic cells. FEBSLett, 1996. 397: p. 269 -272.

40. Meagher LC, S.J., Baker A and Haslett C, Phagocytosis of apoptotic neutrophils does not induce macrophage release of Thromboxane B2. J. Leuk. Biol, 1992. 52: p. 269-273.

41. Savill J, R.Y., Stern M and Haslett C Uptake of post-apoptotic granulocytes via a novel polyanion-inhibitable recognitionmechanism. J.Am.Soc.Nephrol, 1996. 7 p. 17-19.

42. Hughes J, L., VanDamme J andSavill J, Human glomerularmesangial cell phagocytosis of apoptotic neutrophils: Mediation by a novel CD36-independent vitronectin receptor/thrombospondin recognition mechanism that is uncoupled from chemokine secretion. J. Immunol, 1997. 158: p. 4389-4397.

43. Fadok VA, B.D., Konowal A, Freed PW,Westcott JY and Henson PM, Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGFb, PGE2 and PAF. J. Clin. Invest., 1998. 101: p. 890-898.

44. Voll RE, H.M., Roth EA, Stach C and Kalden JR, Immunosuppressive effects of apoptotic cells. Nature, 1997. 390:: p. 350-351.

45. JJ, S.C.a.S., T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature, 1994. 372: p. 100-103.

46. Majno, G., and Joris, I, Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995. 146: p. 3-15.

47. Levin, S., Bucci, T. J., Cohen, S. M., Fix, A. S., Hardisty, J. F., LeGrand, E. K., Maronpot, R. R., and Trump, B. F, The nomenclature of cell death: recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists. Toxicol Pathol, 1999. 27: p. 484-490.

48. Zeiss, C.J., The apoptosis-necrosis continuum: insights from genetically altered mice. Vet Pathol 2003. 40: p. 481-495.

49. Majno, G., and Joris, I, Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995. 146: p. 3-15.

50. Trump, B.F., Berezesky, I. K., Chang, S. H., and Phelps, P. C, The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol, 1997. 25 p. 82-88.

51. Angelillo-Scherrer, L.B.P.F.B.R.K.A., Cell-derived microparticles in haemostasis and vascular medicine. J Thromb Haemost, 2009. 101: p. 439-451.

52. Beyer C, P.D., The role of microparticles in the pathogenesis of rheumatic diseases. Nat Rev Rheumatol, 2010. 6: p. 21-29.

53. JM, F., Cellular microparticles: what are they bad or good for? J Thromb Haemost, 2003. 1: p. 1655-1662.

54. Fox JEB, A.C., Boyles JK, Role of the membrane skeleton in preventing the shedding of procoagulant- rich microvesicles from the platelet plasma membrane. J Cell Biol 1990. 111: p. 483-493.

55. Fox JE, A.C., Reynolds CC, Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets. J BiolChem 1991. 266: p. 13289-13295.

56. McLaughlin PJ, G.J., Mannherz HG, Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature, 1993. 364: p. 685-692.

57. Gasser O, S.J., Activated polymorphonuclear neutrophils siddeminate anti-inflammatory microparticles by ectocytosis. Blood, 2004. 104: p. 2543-2548.

58. Christian B, D.S., The role of microparticles in the pathogenesis of rheumatic disease. Nature, 2010. 6: p. 21-29.

59. Gerke V, M.S., Annexins: from structure to function Physiol Rev, 2002. 82: p. 331-371.

60. Gerke, V., Creutz, C. E. &; Moss, S. E., Annexins: linking Ca2+ signalling to membrane dynamics. . Nature Rev Mol. Cell Biol, 2005. 6: p. 449-461.
61. Rhen, T.C., J. A, Antiinflammatory action of glucocorticoids - new mechanisms for old drugs. N. Engl. J. Med, 2005. 353: p. 1711-1723

62. Parente, L.S., E., Annexin 1: more than an antiphospholipase protein. . Inflamm. Res, 2004. 53: p. 125-132.

63. Perretti, M., Annexin I is stored within gelatinase granules of human neutrophils and mobilised on the cell surface upon adhesion but not phagocytosis. Cell Biol. Int. , 2000. 24: p. 163-174.

64. Wein, S.e.a., Mediation of annexin 1 secretion by a probenecid sensitive ABC transporter in rat inflamed mucosa. Biochem. Pharmacol, 2004. 67: p. 1195-1202.

65. Solito, E.e.a., Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface. . FASEB J, 2006. 20: p. 1498-1500.

66. Walther, A., Riehemann, K. &; Gerke, V. , A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell 2000. 5: p. 831-840.

67. Davidson J, F.R., Milton AS, Peer SH, Rotondo D, Antipyretic actions of human recombinant lipocortin-1 Br J Pharmacol, 1991. 102: p. 7-9.

68. Ferriar SH, C.F., Lorenzetti BB, Michelin MA, Perretti M, Flower RJ, Pools S, Role of lipocortin-1 in the anti-hyperalgesic actions of dexamethasone. Biochem. Pharmacol, 1997. 50: p. 883-888.

69. Ramirez, F., Glucocorticoids induce a Th2 response in vitro. Dev. Immunol., 1998. 6: p. 233-243

70. Ramirez, F., Fowell, D. J., Puklavec, M., Simmonds, S.,Mason, D, Glucocorticoids promote a TH2 cytokine response by CD4+ T cells in vitro. J. Immunol. , 1996. 156: p. 2406-2412.

71. Perretti, M., Dalli J, Exploiting the Annexin A1 pathway for the development of novel anti-inflammatory therapeutics. Br J Pharmacol, 2009. 158: p. 936-946.

72. Lim LH, P.S., Annexin A1: the new face of an old molecule. FASEB J, 2007. 21: p. 968-975.

73. Charles N Serhan, J.S., Resolution of inflammation: the beginning programs the end. Nature Immunology, 2005. 6: p. 1191 - 1197.

74. Dalli J, N.L., Renshaw D, Cooper D, Leung KY, Perretti M, Annexin A1 mediates the rapid anti-inflammatory effects of neutrophil- derived microparticles. Blood, 2008. 112: p. 2512-2519.

75. R. Mangiarotti, M.D., R. Alberici, and C. Pellicciari, All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells. Br J Cancer, 1998. 77: p. 186-191. .

76. 吳虹毅, Role of lipoxin A4 in resolution phase of retinoic acid syndrome 國立陽明大學生理學研究所碩士論文, 2009.

77. Du, C., Redner, R. L., Cooke, M. P. &; Lavau, C, Overexpression of wild type retinoic acid receptor α (RARα) recapitulates retinoic acid sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RAR α fusion genes. Blood, 1999. 94: p. 793-802

78. Zhong, S., Salomoni, P. &; Pandolfi, P. P. , The transcriptional role of PML and the nuclear body. Nature Cell Biol. , 2000. 2: p. 85-90.

79. Cicalese, A.e.a., The tumor suppressor p53 regulates polarity of self‑renewing divisions in mammary stem cells. Cell 2009. 138: p. 1083-1095.

80. Chen, H.d.T.a.Z., Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nature Rev, 2010. 10: p. 775-783.

81. E E Torr, D.H.G., L Thomas, D M Goodall, A Bielemeier, R Willetts, H R Griffiths, L J Marshall and A Devitt, Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells. Cell Death Differ , 2012. 19: p. 671-679.
82. Irini Bournazou, J.D.P., Rodger Duffin, Stylianos Bournazos, Lynsey A. Melville, Simon B. Brown, Adriano G. Rossi, and Christopher D. Gregory, Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin. J Clin Invest, 2009. 119: p. 20-32.

83. Danute Pupjalis, J.G., Diane J. Kottas, Volker Gerke, Ursula Rescher, Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling. EMBO Molecular Medicine, 2011. 3: p. 102-114.

84. 簡弘育, Annexin A1 mediateds the anti-inflammatory effect of microparticles released from retinoic acid- treated acute promyelocytic leukemic cells. 國立陽明大學 生理學研究所碩士論文, 2010.