臺灣博碩士論文加值系統

過敏性氣喘是由Th2細胞分泌細胞激素IL-4、IL-5及IL-13所引起的呼吸道慢性發炎，會造成肺部中嗜酸性白血球聚集，IgE抗體的產生及肥大細胞去顆粒化釋放出組織胺，導致呼吸道平滑肌收縮，黏液分泌增多而產生呼吸困難的現象。本篇研究的冬凌草甲素 (Oridonin) 是從冬凌草中分離出來的四環二萜類化合物，具有抗發炎、抗癌及抗菌的功能。而目前對於冬凌草甲素在過敏性疾病的研究了解甚少；因此我們想探討冬凌草甲素是否能透過抗發炎的特性，對參與過敏性氣喘疾病生成的免疫細胞進行調節，以達到改善過敏性氣喘症狀的效果。首先利用體外培養的小鼠骨髓衍生性樹突細胞，在LPS刺激下與冬凌草甲素共同培養，實驗結果發現，樹突細胞之IL-12及IL-10分泌量皆有顯著降低，而前發炎激素IL-6分泌量也有顯著減少。不過冬凌草甲素對於樹突細胞上的表面分子MHC class II、CD40、CD80、CD86、PD-L1和CD54的表現則不具影響。而這樣的樹突細胞亦無法抑制CD4+ T細胞的增殖與細胞激素的產生。但是若將冬凌草甲素直接作用於經anti-CD3/CD28抗體活化的CD4+ T細胞上，則發現冬凌草甲素對於活化的CD4+ T細胞的細胞激素的產生有明顯的抑制作用，顯示冬凌草甲素可能具有抑制T細胞活化的功能。接著將冬凌草甲素應用在治療過敏性氣喘小鼠動物模式上，實驗結果顯示冬凌草甲素可以有效的減輕小鼠呼吸道阻力的上升與肺部的發炎反應現象，也能抑制經過敏原OVA刺激後脾臟細胞的Th2 細胞激素 IL-4、IL-5和IL-13 的分泌；另外能降低全身血清中 OVA 專一性抗體 IgE 和 IgG1 的表現量，此外也可觀察到冬凌草甲素能促使小鼠體內表現 CD25+Foxp3+ CD4+之調節型T 細胞(Treg)的數目增加。綜合以上實驗結果推測，冬凌草甲素對免疫細胞調控的途徑可能是藉由增加 CD25+Foxp3+ CD4+的 Treg 細胞數目，以達到抑制 Th2 細胞反應的效果，使過敏性氣喘的症狀獲得改善。因此，我們認為未來或許也能開發冬凌草甲素成為治療過敏性氣喘之藥物。

Allergic asthma is a chronic inflammatory lung disease of the airway, characterized by airway eosinophil accumulation, goblet cell hyperplasia with mucus hypersecretion, and hyper-responsiveness in response to inhaled allergens. The inflammatory process in allergic asthma is dominated by type 2 T helper (Th2) cells that produce interleukin-5 (IL-5), IL-4 and IL-13, which activate eosinophil and induce immunoglobulin E(IgE) production by B cells.. Oridonin, an ent-kaurane diterpenoid compound (C20H28O6), is isolated from the traditional Chinese herb Rabdosia rubescens. Oridonin has shown anti-tumor, anti-bacterial, and anti-inflammatory properties. Thus, we want to investigate the anti-allergic effects and mechanisms of Oridonin on immune effector cells and ovalbumin(OVA)-induced asthmatic mice. Our data showed that Oridonin inhibited the production of IL-12, IL-10 and IL-6 in LPS-stimulated DCs.However,Oridonin treatment didn’t effect the expression of MHC class II、CD40、CD80、CD86、PD-L1 and CD54 molercules in LPS-stimulated DCs. By mix lymphocyte reaction, such Oridonin plus LPS-treated DCs didn’t inhibit T cells proliferation and cytokine production. On the other hand, we found that Oridonin could directly suppress anti-CD3/CD28 antibodies-activated CD4+ T cells cytokine production. Based on the above results, we though that Oridonin may have the therapeutic ability to reduce allergic-specific immune responses in vivo. In therapeutic animal model of OVA-induced asthma, Oridonin treatment significantly attenuated the severe levels of airway hyper-responsiveness and the lung inflammation, as well as Th2 cytokines secretion in OVA-stimulated splenocytes. Additionally, the expression levels of OVA-specific IgE and IgG1 were reduced in these Oridonin-treated mice. Notably, the administration of Oridonin in enhanced the generation of CD25+ Foxp3+ CD4+ regulatort T cells (Treg). These findings indicated that Oridonin may inhibit the development of allergic asthma by increasing the number of CD25+ Foxp3+ CD4+ Treg cells in these mice. In the future, we hope that Oridonin can be development as a novel agent to treat allergic asthma.

第一章 緒論 ………………………………………………………………………1
1. 過敏性氣喘簡介 ………………………………………………………………...2
1.1過敏性氣喘之致病機轉………………………………………………………....2
1.2 樹突狀細胞 ………………………………………………………………….....3
1.3 T細胞的分類與功能…………………………………………………………....4
1.4 過敏性氣喘之治療………………………………………………………….......5
1.5. 冬凌草甲素 (Oridonin) 介紹……………………………………………….....6
第二章 探討冬凌草甲素對於樹突細胞與CD4+T細胞免疫調控的影響..……...9
2.1研究動機與目的………………………………………………………………...10
2.2研究方法與材料………………………………………………………………...11
2.3研究結果………………………………………………………………...............17
第三章 探討冬凌草甲素對於OVA所誘發之氣喘動物模式治療的效果..……..20
3.1研究動機與目的………………………………………………………………...21
3.2研究方法與材料………………………………………………………………...22
3.3研究結果………………………………………………………………...............27
第四章 討論………………………………………………………………...............30
4.1. 冬凌草甲素對樹突狀細胞免疫調控之影響……….........................................31
4.2. 冬凌草甲素對T細胞免疫調控之影響……………………………………….32
4.3. 冬凌草甲素對OVA誘導氣喘動物模式之影響……………………………...32
第五章 結論與未來方向………............... ………............... ………............... ……35
第六章 圖………………………………………………………………....................37
參考文獻……………………………………………………………………………..61

1. Gallelli L, Busceti MT, Vatrella A, Maselli R, Pelaia G (2013) Update on anticytokine treatment for asthma. Biomed Res Int 2013: 104315.
2. Hall S, Agrawal DK (2014) Key mediators in the immunopathogenesis of allergic asthma. Int Immunopharmacol.
3. Kim HY, DeKruyff RH, Umetsu DT (2010) The many paths to asthma: phenotype shaped by innate and adaptive immunity. Nat Immunol 11: 577-584.
4. Masoli M, Fabian D, Holt S, Beasley R, Global Initiative for Asthma P (2004) The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy 59: 469-478.
5. Kumar RK, Foster PS (2002) Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 27: 267-272.
6. Amin K (2012) The role of mast cells in allergic inflammation. Respir Med 106: 9-14.
7. Galli SJ, Tsai M (2012) IgE and mast cells in allergic disease. Nat Med 18: 693-704.
8. Stone KD, Prussin C, Metcalfe DD (2010) IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol 125: S73-80.
9. Holgate ST (2012) Innate and adaptive immune responses in asthma. Nat Med 18: 673-683.
10. Deckers J, Branco Madeira F, Hammad H (2013) Innate immune cells in asthma. Trends Immunol 34: 540-547.
11. Bandeira-Melo C, Bozza PT, Weller PF (2002) The cellular biology of eosinophil eicosanoid formation and function. J Allergy Clin Immunol 109: 393-400.
12. Gaurav R, Agrawal DK (2013) Clinical view on the importance of dendritic cells in asthma. Expert Rev Clin Immunol 9: 899-919.
13. van Helden MJ, Lambrecht BN (2013) Dendritic cells in asthma. Curr Opin Immunol 25: 745-754.
14. Nakano H, Free ME, Whitehead GS, Maruoka S, Wilson RH, et al. (2012) Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens. Mucosal Immunol 5: 53-65.
15. Steinman RM, Cohn ZA (1974) Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro. J Exp Med 139: 380-397.
16. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392: 245-252.
17. Mellman I, Steinman RM (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106: 255-258.
18. Steinman RM (1991) The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9: 271-296.
19. Lambrecht BN, Hammad H (2010) The role of dendritic and epithelial cells as master regulators of allergic airway inflammation. Lancet 376: 835-843.
20. Trinchieri G, Santoli D, Dee RR, Knowles BB (1978) Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Identification of the anti-viral activity as interferon and characterization of the human effector lymphocyte subpopulation. J Exp Med 147: 1299-1313.
21. Belz GT, Nutt SL (2012) Transcriptional programming of the dendritic cell network. Nat Rev Immunol 12: 101-113.
22. Hubert FX, Voisine C, Louvet C, Heslan M, Josien R (2004) Rat plasmacytoid dendritic cells are an abundant subset of MHC class II+ CD4+CD11b-OX62- and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG. J Immunol 172: 7485-7494.
23. Mbongue J, Nicholas D, Firek A, Langridge W (2014) The role of dendritic cells in tissue-specific autoimmunity. J Immunol Res 2014: 857143.
24. van Ree R, Hummelshøj L, Plantinga M, Poulsen LK, Swindle E (2014) Allergic sensitization: host-immune factors. Clin Transl Allergy 4: 12.
25. Steinman L (2007) A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 13: 139-145.
26. Park H, Li Z, Yang XO, Chang SH, Nurieva R, et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6: 1133-1141.
27. Bettelli E, Korn T, Kuchroo VK (2007) Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 19: 652-657.
28. Asseman C, Mauze S, Leach MW, Coffman RL, Powrie F (1999) An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J Exp Med 190: 995-1004.
29. Shevach EM, Davidson TS, Huter EN, Dipaolo RA, Andersson J (2008) Role of TGF-Beta in the induction of Foxp3 expression and T regulatory cell function. J Clin Immunol 28: 640-646.
30. Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA (2001) A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol 166: 7282-7289.
31. Umetsu DT, Akbari O, Dekruyff RH (2003) Regulatory T cells control the development of allergic disease and asthma. J Allergy Clin Immunol 112: 480-487; quiz 488.
32. Lowe PJ, Renard D (2011) Omalizumab decreases IgE production in patients with allergic (IgE-mediated) asthma; PKPD analysis of a biomarker, total IgE. Br J Clin Pharmacol 72: 306-320.
33. Holgate ST (2011) Pathophysiology of asthma: what has our current understanding taught us about new therapeutic approaches? J Allergy Clin Immunol 128: 495-505.
34. Pelaia G, Vatrella A, Gallelli L, Renda T, Cazzola M, et al. (2006) Respiratory infections and asthma. Respir Med 100: 775-784.
35. Gauvreau GM, Boulet LP, Cockcroft DW, Fitzgerald JM, Carlsten C, et al. (2011) Effects of interleukin-13 blockade on allergen-induced airway responses in mild atopic asthma. Am J Respir Crit Care Med 183: 1007-1014.
36. Oh CK, Raible D, Geba GP, Molfino NA (2011) Biology of the interleukin-9 pathway and its therapeutic potential for the treatment of asthma. Inflamm Allergy Drug Targets 10: 180-186.
37. Cheng G, Arima M, Honda K, Hirata H, Eda F, et al. (2002) Anti-interleukin-9 antibody treatment inhibits airway inflammation and hyperreactivity in mouse asthma model. Am J Respir Crit Care Med 166: 409-416.
38. Parker JM, Oh CK, LaForce C, Miller SD, Pearlman DS, et al. (2011) Safety profile and clinical activity of multiple subcutaneous doses of MEDI-528, a humanized anti-interleukin-9 monoclonal antibody, in two randomized phase 2a studies in subjects with asthma. BMC Pulm Med 11: 14.
39. Wu YX, Zhang W, Li JC, Liu N (2012) Chemical constituents of flowers and fruits of Rabdosia excisa. Chin J Nat Med 10: 43-47.
40. Fujita E, Nagao Y, Node M, Kaneko K, Nakazawa S, et al. (1976) Antitumor activity of the Isodon diterpenoids: structural requirements for the activity. Experientia 32: 203-206.
41. Guo W, Zheng P, Zhang J, Ming L, Zhou C, et al. (2013) Oridonin suppresses transplant rejection by depleting T cells from the periphery. Int Immunopharmacol 17: 1148-1154.
42. Liu JJ, Wu XY, Peng J, Pan XL, Lu HL (2004) Antiproliferation effects of oridonin on HL-60 cells. Ann Hematol 83: 691-695.
43. Zhou L, Sun L, Wu H, Zhang L, Chen M, et al. (2013) Oridonin ameliorates lupus-like symptoms of MRL(lpr/lpr) mice by inhibition of B-cell activating factor (BAFF). Eur J Pharmacol 715: 230-237.
44. Ku CM, Lin JY (2013) Anti-inflammatory effects of 27 selected terpenoid compounds tested through modulating Th1/Th2 cytokine secretion profiles using murine primary splenocytes. Food Chem 141: 1104-1113.
45. Xu Y, Xue Y, Wang Y, Feng D, Lin S, et al. (2009) Multiple-modulation effects of Oridonin on the production of proinflammatory cytokines and neurotrophic factors in LPS-activated microglia. Int Immunopharmacol 9: 360-365.
46. Hu AP, Du JM, Li JY, Liu JW (2008) Oridonin promotes CD4+/CD25+ Treg differentiation, modulates Th1/Th2 balance and induces HO-1 in rat splenic lymphocytes. Inflamm Res 57: 163-170.
47. Cui Q, Tashiro S, Onodera S, Ikejima T (2006) Augmentation of oridonin-induced apoptosis observed with reduced autophagy. J Pharmacol Sci 101: 230-239.
48. Li CY, Wang EQ, Cheng Y, Bao JK (2011) Oridonin: An active diterpenoid targeting cell cycle arrest, apoptotic and autophagic pathways for cancer therapeutics. Int J Biochem Cell Biol 43: 701-704.
49. Zhang X, Chen LX, Ouyang L, Cheng Y, Liu B (2012) Plant natural compounds: targeting pathways of autophagy as anti-cancer therapeutic agents. Cell Prolif 45: 466-476.
50. Hu HZ, Yang YB, Xu XD, Shen HW, Shu YM, et al. (2007) Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line. Acta Pharmacol Sin 28: 1819-1826.
51. KUBO I, TANIGUCHI M, SATOMURA Y, KUBOTA T (1974) Antibacterial activity and chemical structure of diterpenoids. Agricultural and Biological Chemistry 38: 1261-1262.
52. 刘净, 梁敬钰, 谢韬 (2004) 冬凌草研究进展. 海峡药学 16: 1-7.
53. Padrid PA, Mathur M, Li X, Herrmann K, Qin Y, et al. (1998) CTLA4Ig inhibits airway eosinophilia and hyperresponsiveness by regulating the development of Th1/Th2 subsets in a murine model of asthma. Am J Respir Cell Mol Biol 18: 453-462.
54. Ray A, Cohn L (2000) Altering the Th1/Th2 balance as a therapeutic strategy in asthmatic diseases. Curr Opin Investig Drugs 1: 442-448.
55. Park HJ, Lee CM, Jung ID, Lee JS, Jeong YI, et al. (2009) Quercetin regulates Th1/Th2 balance in a murine model of asthma. Int Immunopharmacol 9: 261-267.
56. Moser M, Murphy KM (2000) Dendritic cell regulation of TH1-TH2 development. Nat Immunol 1: 199-205.
57. Liu YJ, Kanzler H, Soumelis V, Gilliet M (2001) Dendritic cell lineage, plasticity and cross-regulation. Nat Immunol 2: 585-589.
58. Deo SS, Mistry KJ, Kakade AM, Niphadkar PV (2010) Role played by Th2 type cytokines in IgE mediated allergy and asthma. Lung India 27: 66-71.
59. Mazzarella G, Bianco A, Catena E, De Palma R, Abbate GF (2000) Th1/Th2 lymphocyte polarization in asthma. Allergy 55 Suppl 61: 6-9.
60. Akdis CA (2012) Therapies for allergic inflammation: refining strategies to induce tolerance. Nat Med 18: 736-749.
61. Snapper CM, Peschel C, Paul WE (1988) IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide. J Immunol 140: 2121-2127.
62. Wenzel SE (2012) Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med 18: 716-725.