臺灣博碩士論文加值系統

慢性阻塞性肺病係ㄧ種因慢性支氣管炎或肺氣腫所引發，而導致呼吸道氣流阻塞為主要特徵的肺部疾病。其發病率與死亡率很高，預計到2020年時，其死因排名將會升至第五位。雖然已有許多COPD的致病因子被鑑定出來，目前讓肺部及支氣管持續發炎的機制仍不清楚。現今自體免疫反應已被證實參與於COPD致病機轉之中，但是尚未發現和COPD有關的自體抗原和自體抗體。本文中，我們利用西方墨點法分析COPD患者的血清並證實其血清內存在著對抗肺泡上皮細胞抗原的自體抗體。我們發現確實有對抗肺泡上皮細胞抗原的自體抗體存在於患者血清中，其最主要相對應的抗原分子量為98, 86, 75, 66, 51, 45, 和31(kD)。利用免疫沉澱法和質譜儀分析45-kD位置的抗原為人類細胞角質蛋白質18(human cytokeratin 18)。我們發現患者和控制組血清中抗細胞角質蛋白質18自體抗體的盛行率分別為74%和32%。使用酵素免疫分析法來偵測抗細胞角質蛋白質18自體抗體的量發現在患者血清中也遠高於控制組血清，而主要病灶為肺氣腫亦高於病灶為慢性支氣管炎的族群。雖然抗細胞角質蛋白質18自體抗體的量與FEV1 (L)和FEV1 (% 預測值)有相關性，卻和疾病分期並無關聯。這是第一次證實與COPD相關的人類細胞角質蛋白質18自體抗原的存在。

Chronic obstructive pulmonary disease ( COPD ) is characterized by airflow obstruction due to chronic bronchitis or pulmonary emphysema。The morbidity and mortality of COPD is related high. Moreover it is expected to rise from its current position as the fifth most common cause of death，worldwide, by the year 2020。Although some risk factors for COPD have been identified，the mechanism of persistent chronic airway inflammation remains unclear。The involvement of autoimmune responses in the pathogenesis of COPD has been proposed; however，no autoantigens or corresponding autoantibodies associated with COPD have been identified. Here，we analyzed the sera of COPD patients to investigate the possible existence of circulating autoantibodies against the A549 cellular proteins。We showed the existence of autoantibodies against the pulmonary cellular antigens in the sera of COPD patients，and the molecular weights (kD) of the predominant immunoreactive autoantigens were 98，86，75，66，51，45 and 31， respectively。Immunoprecipitation and MALDI-TOF mass spectrometry revealed that the 45-kD target autoantigen was human cytokeratin 18 protein。We showed that the prevalence of anti-cytokeratin 18 autoantibodies in the sera of COPD patients and controls was 74% and 32%, respectively。We used ELISA to detect anti-cytokeratin 18 autoantibody level and also found that the anti-cytokeratin 18 autoantibody level was significantly higher in the COPD group than in the control group and in the predominantly emphysema group than in the predominantly chronic bronchitis group。Although we showed that the anti-cytokeratin 18 autoantibody level was significantly associated with FEV1 (L) and FEV1 (% pred)，the presence of the anti-cytokeratin 18 autoantibody was not associated with the GOLD stage of the disease.。This is the first report on the identification of the pulmonary epithelial cytokeratin 18 protein as an autoantigen associated with COPD and the demonstration of the prevalence of anti-cytokeratin 18 autoantibodies in COPD patients。

指導教授推薦書
口試委員會審定書
長庚大學博碩士紙本論文著作授權書
國家圖書館書授權書.……………...…………………………………...iii
長庚大學授書…...….…………………………………………………...iv
誌謝….……………………..…………………………………………….v
中文摘要……………………..........…………………………………….vi
英文摘要…………………....……….........…………………………….vii
目錄………………..…………………………………………………….iv
圖表目錄……...……………………………………………...…………xii
第一章 簡介………………………………………………………...….1
1.1 慢性阻塞性肺病定義……………………………………………...1
1.2 慢性阻塞性肺病診斷及分期…………………………………..….3
1.3 慢性阻塞性肺病的致病機制……………………………..……….4
1.4 自體免疫假說………………………………………………...........7
第二章 研究目的…………………………………................................12
第三章 材料…………………………………………………………....13
3.1 細胞株及其培養基……………………...………………………....13
3.2 蛋白質定量………………………………….………………...…...13
3.3 蛋白質電泳……………………………………….………………..13
3.4 西方墨點法…………………………………………….…………..14
3.5 銀染…………………………………………….......……………....15
3.6 免疫沉澱法…………………….………………………………......15
第四章 方法………………………………………………….………...16
4.1 細胞培養………………………….……………………………......16
4.2 細胞蛋白質萃取..…………………………...……………...……...16
4.4 蛋白質濃度定量..……………………………………...…...……...16
4.5 蛋白質電泳………………………………………………......….....17
4.6 Coomassie Blue stain …………………...…………………......…...17
4.7 西方點墨法………………………………………………………...17
4.8 免疫沉澱法…………………………………………………....…...18
4.9 銀染……..……………………………………………......………...18
4.10 酵素免疫分析法……………..…………………...………..……..19
4.11 組織檢體與血清…………………..…………………........……...19
4.12 統計分析……………………………………………………..…...19
第五章 結果……………………………………………………………21
5.1篩檢COPD患者血清中是否存在有抗肺臟細胞之自體抗體…......21
5.2分析自體抗體效價與COPD臨床病程的關係………………….....21
第六章 討論…………………………………………………….....…...26
參考文獻…………………………………………………...........……...30































圖表目錄
圖一 western-blot assay偵測研究樣本血清中的抗肺臟細胞蛋白的自體抗體……………………………………………………..…...……….35
圖二 免疫沉澱後免疫複體以12%SDS-PAGE膠分離後之銀染結果.36
圖三 西方墨點法鑑定與篩檢研究樣本血清中之Anti-recombinant human cytokeratin 18 protein自體抗體………………………..………37
圖四 確認尋找ELISA條件之檢體………………………………...….38
圖五 抗原吸附濃度篩選………………………………………...…….39
圖六 血清及二級抗體稀釋倍數篩選……………………………..…..40
圖七 呈色時間篩選………………………………………………..…..41
圖八 西方墨點法比較COPD患者與控制組間自體抗體量的相關性.42
圖九 利用酵素免疫分析法比較COPD患者與控制組間自體抗體量的相關性……………………………………………………..……………43
圖十 利用西方墨點法比較肺氣腫與慢性支氣管炎之COPD患者間Anti-cytokeratin 18 autoantibody 量的相關性………………………...44
圖十一 利用酵素免疫分析法比較肺氣腫與慢性支氣管炎之COPD患者間Anti-cytokeratin 18 autoantibody 量的相關性……………..……45
圖十二 Receiver Operating Characteristics Curve ( ROC curve)分析比較西方墨點法及酵素免疫分析法.……………………………………..46

參考文獻
1. Disease, t.G.i.f.c.O.L., http://www.goldcopd.com/.
2. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society. Am J Respir Crit Care Med, 1995. 152(5 Pt 2): p. S77-121.
3. The definition of emphysema. Report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases workshop. Am Rev Respir Dis, 1985. 132(1): p. 182-5.
4. Organization, W.H., http://www.who.int.
5. 行政院衛生署統計網, http://www.doh.gov.tw/statistic.
6. AS., B., Risk factors for COPD. Eur Respir Rev, 1996. 6: p. 253-8.
7. 台灣胸腔暨重症加護醫學會, 慢性阻塞性肺病診治指引.
8. Samet, J.M. and P. Lange, Longitudinal studies of active and passive smoking. Am J Respir Crit Care Med, 1996. 154(6 Pt 2): p. S257-65.
9. Shapiro, S.D. and E.P. Ingenito, The pathogenesis of chronic obstructive pulmonary disease: advances in the past 100 years. Am J Respir Cell Mol Biol, 2005. 32(5): p. 367-72.
10. Shapiro, S.D., Vascular atrophy and VEGFR-2 signaling: old theories of pulmonary emphysema meet new data. J Clin Invest, 2000. 106(11): p. 1309-10.
11. Laurell CB, E.S., The electrophoretic alpha-globulin pattern of serum in alpha-antitrypsin deficiency. Scand J Clin Lab Invest, 1963. 15: p. 132–140.
12. Gross P, P.E., Tolker E, Babyak M, Kaschak M, Experimental emphysema: its production with papain in normal and silicotic rats. Arch Environ Health, 1965. 11: p. 50–58.
13. Evans, M.D. and W.A. Pryor, Cigarette smoking, emphysema, and damage to alpha 1-proteinase inhibitor. Am J Physiol, 1994. 266(6 Pt 1): p. L593-611.
14. Caughey, G.H., Serine proteinases of mast cell and leukocyte granules. A league of their own. Am J Respir Crit Care Med, 1994. 150(6 Pt 2): p. S138-42.
15. MacNee, W. and I. Rahman, Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease? Trends Mol Med, 2001. 7(2): p. 55-62.
16. Lannan, S., K. Donaldson, D. Brown, and W. MacNee, Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro. Am. J. Physiol, 1994. 266: p. L92–L100.
17. Hautamaki, R. D., D. K. Kobayashi, R. M. Senior and S. D. Shapiro (1997). "Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice." Science 277(5334): 2002-4.
18. Grumelli, S., D. B. Corry, L. Z. Song, L. Song, L. Green, J. Huh, J. Hacken, R. Espada, R. Bag, D. E. Lewis and F. Kheradmand (2004). "An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema." PLoS Med 1(1): e8.
19. Zheng, T., Z. Zhu, Z. Wang, R. J. Homer, B. Ma, R. J. Riese, Jr., H. A. Chapman, Jr., S. D. Shapiro and J. A. Elias (2000). "Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema." J Clin Invest 106(9): 1081-93.
20. Wang, Z., T. Zheng, Z. Zhu, R. J. Homer, R. J. Riese, H. A. Chapman, Jr., S. D. Shapiro and J. A. Elias (2000). "Interferon gamma induction of pulmonary emphysema in the adult murine lung." J Exp Med 192(11): 1587-600.
21. Kasahara, Y., R. M. Tuder, L. Taraseviciene-Stewart, T. D. Le Cras, S. Abman, P. K. Hirth, J. Waltenberger and N. F. Voelkel (2000). "Inhibition of VEGF receptors causes lung cell apoptosis and emphysema." J Clin Invest 106(11): 1311-9.
22. Aoshiba, K., N. Yokohori, and A. Nagai, Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol, 2003. 28(5): p. 555-62.
23. Birring, S.S., et al., Clinical, radiologic, and induced sputum features of chronic obstructive pulmonary disease in nonsmokers: a descriptive study. Am J Respir Crit Care Med, 2002. 166(8): p. 1078-83.
24. Aaron, S. D., J. B. Angel, M. Lunau, K. Wright, C. Fex, N. Le Saux and R. E. Dales (2001). "Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease." Am J Respir Crit Care Med 163(2): 349-55.
25. Agusti, W.M.e.a., Hypothesis: Does COPD have an autoimmune component ? Thorax, 2003. 58(10): p. 832-834.
26. Petty, T.L., The history of COPD. Int J Chron Obstruct Pulmon Dis, 2006. 1(1): p. 3-14.
27. Hogg, J.C., Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. Lancet, 2004. 364(9435): p. 709-21.
28. Hodge, S., G. Hodge, R. Scicchitano, P. N. Reynolds and M. Holmes (2003). "Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells." Immunol Cell Biol 81(4): 289-96.
29. Kuwano, K., N. Hagimoto, M. Kawasaki, T. Yatomi, N. Nakamura, S. Nagata, T. Suda, R. Kunitake, T. Maeyama, H. Miyazaki and N. Hara (1999). "Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis." J Clin Invest 104(1): 13-9.
30. O'Garra, A. and P. Vieira, Regulatory T cells and mechanisms of immune system control. Nat Med, 2004. 10(8): p. 801-5.
31. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda and T. Takahashi (2001). "Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance." Immunol Rev 182: 18-32.
32. Lee, S. H., S. Goswami, A. Grudo, L. Z. Song, V. Bandi, S. Goodnight-White, L. Green, J. Hacken-Bitar, J. Huh, F. Bakaeen, H. O. Coxson, S. Cogswell, C. Storness-Bliss, D. B. Corry and F. Kheradmand (2007). "Antielastin autoimmunity in tobacco smoking-induced emphysema." Nat Med 13(5): 567-9.
33. Fontenot, J.D. and A.Y. Rudensky, A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol, 2005. 6(4): p. 331-7.
34. Taraseviciene-Stewart, L., I. S. Douglas, P. S. Nana-Sinkam, J. D. Lee, R. M. Tuder, M. R. Nicolls and N. F. Voelkel (2006). "Is alveolar destruction and emphysema in chronic obstructive pulmonary disease an immune disease?" Proc Am Thorac Soc 3(8): 687-90.
35. Taraseviciene-Stewart, L., R. Scerbavicius, K. H. Choe, M. Moore, A. Sullivan, M. R. Nicolls, A. P. Fontenot, R. M. Tuder and N. F. Voelkel (2005). "An animal model of autoimmune emphysema." Am J Respir Crit Care Med 171(7): 734-42.
36. Marrack, P., J. Kappler, and B.L. Kotzin, Autoimmune disease: why and where it occurs. Nat Med, 2001. 7(8): p. 899-905.
37. Ermann, J. and C.G. Fathman, Autoimmune diseases: genes, bugs and failed regulation. Nat Immunol, 2001. 2(9): p. 759-61.
38. Moll, R., W. W. Franke, D. L. Schiller, B. Geiger and R. Krepler (1982). "The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells." Cell 31(1): 11-24.
39. Dobashi, N., J. Fujita, M. Murota, Y. Ohtsuki, I. Yamadori, T. Yoshinouchi, R. Ueda, S. Bandoh, T. Kamei, M. Nishioka, T. Ishida and J. Takahara (2000). "Elevation of anti-cytokeratin 18 antibody and circulating cytokeratin 18: anti-cytokeratin 18 antibody immune complexes in sera of patients with idiopathic pulmonary fibrosis." Lung 178(3): 171-9.
40. Nahm, D. H., Y. E. Lee, E. J. Yim, H. S. Park, H. Yim, Y. Kang and J. K. Kim (2002). "Identification of cytokeratin 18 as a bronchial epithelial autoantigen associated with nonallergic asthma." Am J Respir Crit Care Med 165(11): 1536-9.
41. Murota, M., M. Nishioka, J. Fujita, N. Dobashi, F. Wu, Y. Ohtsuki, S. Hojo, J. Takahara and S. Kuriyama (2001). "Anti-cytokeratin antibodies in sera of the patients with autoimmune hepatitis." Clin Exp Immunol 125(2): 291-9.
42. Borg, A.A., P.T. Dawes, and D.L. Mattey, Increased levels of IgA antibodies to cytokeratin-18 and epidermal keratin in rheumatoid arthritis. Arthritis Rheum, 1993. 36(2): p. 229-33.
43. Kamradt, T. and N.A. Mitchison, Tolerance and autoimmunity. N Engl J Med, 2001. 344(9): p. 655-64.
44. Albert, L.J. and R.D. Inman, Molecular mimicry and autoimmunity. N Engl J Med, 1999. 341(27): p. 2068-74.
45. Leers, M. P., W. Kolgen, V. Bjorklund, T. Bergman, G. Tribbick, B. Persson, P. Bjorklund, F. C. Ramaekers, B. Bjorklund, M. Nap, H. Jornvall and B. Schutte (1999). "Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis." J Pathol 187(5): 567-72.
46. Caulin, C., G.S. Salvesen, and R.G. Oshima, Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol, 1997. 138(6): p. 1379-94.
47. Krysko, D.V., K. D'Herde, and P. Vandenabeele, Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis, 2006. 11(10): p. 1709-26.
48. Cline, A.M. and M.Z. Radic, Apoptosis, subcellular particles, and autoimmunity. Clin Immunol, 2004. 112(2): p. 175-82.
49. Feghali-Bostwick, C. A., A. S. Gadgil, L. E. Otterbein, J. M. Pilewski, M. W. Stoner, E. Csizmadia, Y. Zhang, F. C. Sciurba and S. R. Duncan (2008). "Autoantibodies in patients with chronic obstructive pulmonary disease." Am J Respir Crit Care Med 177(2): 156-63.
50. Agusti, A., Thomas a. Neff lecture. Chronic obstructive pulmonary disease: a systemic disease. Proc Am Thorac Soc, 2006. 3(6): p. 478-81.
51. Finkelstein, E.I., M. Nardini, and A. van der Vliet, Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease? Am J Physiol Lung Cell Mol Physiol, 2001. 281(3): p. L732-9.