臺灣博碩士論文加值系統

一個良好生物標記物不但可以幫助醫師對於病人病情判斷、評估及治療，甚至可能帶來龐大的商機。氣喘為一種呼吸道的慢性疾病，這類的疾病以及發炎反應的發生都跟免疫系統有關係，癌症則是目前醫學界仍無法解決的問題。近期的研究發現??-enolase (本篇簡稱ENO1)為一個具有潛力的生物標記，此蛋白及相對應的抗體出現可能可以用來作為這些疾病的偵測。我們蒐集了氣喘病人、具有高腫瘤標記的病人血清，利用基因選殖的方法表現並純化重組人類、金黃色葡萄球菌 (Staphylococcus aureus；本篇簡稱 SA)、肺炎鏈球菌 (Streptococcus pneumoniae；簡稱SP)及白色念珠菌 (Candida albicans；簡稱CA)的???????﹞妨寣A以Western blot與ELISA的方式偵測不同病人血清中抗體的存在情形。結果發現氣喘病人的血液中所帶有的anti-human ENO1、anti-SA ENO1及anti-CA ENO1 IgG抗體量與健康民眾比起來有較高情況，並且達到統計上的意義。我們得到結果發現anti-human ENO1自體抗體存在的情形在有高濃度AFP及CA-199兩種cancer marker的血清做出的結果體內anti-human ENO1自體抗體的濃度比正常組的還要高；anti-SA ENO1抗體存在的情形在有高濃度CA-125及CA-153兩種cancer marker的血清做出的結果體內anti-SA ENO1抗體的濃度比正常組的還要高；anti-SP ENO1抗體的ELISA結果在所有高濃度cancer marker血清病人和正常人血液的吸光值相比皆未達到統計上的差異；anti-CA ENO1抗體存在的情形在有高濃度AFP、CEA及PSA三種cancer marker的血清做出的結果體內anti-SA ENO1抗體的濃度比正常組的還要高。
我們做的研究帶出了一個方向，在未來的研究者可以根據有意義的結果增加受試者的數量來證實我們的結果，或是設計其他的實驗來探討ENO1抗體存在的意義為何。

A meaningful biomarker not only helps clinical doctor to evaluate patient’s condition and treatment, but also brings huge business opportunities. Asthma is a chronic respiratory disease. Asthma and inflammatory reaction are related to the immune system. And cancer is a big problem of medical profession. Recently, alpha-enolase protein (ENO1) and its antibodies had found as a potential biomarker to predict those diseases. We used the recombinant Staphylococcus aureus (SA) Streptococcus pneumoniae (SP) and Candida albicans (CA) ENO1 to analysis the presence of antibodies in patients with asthma and high cancer marker by western blot and ELISA method.
The results showed that patients with severe asthma had higher concentration of anti-human ENO1, anti-SA ENO1 and anti-CA ENO1 IgG antibodies. It also found some results in patients with high cancer marker. Anti-human ENO1 autoantibodies present a high concentration situation in patients with high AFP and CA-199 cancer marker. High anti-SA ENO1 antibodies are observed in patients with high CA-125 and CA-153 cancer marker. And high anti-CA ENO1 antibodies appear in patients with high AFP, CEA and PSA cancer marker. It is special that the concentration of anti-SP ENO1 antibodies is not different between all patients and control group.
In this study, we provide a direction. Researchers can increase the number of subject to further confirm our results in the future. Or they can design new experiments to explain the mechanism of ENO1 antibody causing disease.

目錄………………………………………………………………………I
表目錄…………………………………………………………………..IV
圖目錄…………………………………………………………………...V
中文摘要………………………………………………………………..VI
英文摘要……………………………………………………………...VIII
壹. 前言………………………………………………………………….09
一、 Alpha-enolase簡介………………………………...…………09
二、 氣喘疾病的簡介……………………………………..………12
三、 生物標記的特性……………………………………………..14
貳. 實驗目的…………………………………………………………….19
參. 實驗材料與方法………………..…………………………………...16
一、 實驗材料……………………………………..………………16
二、 實驗方法……………………………………………………..20
1. 病人血清蒐集………………………………………………20
2. ???????○J白質的製備………………………………………..21
3. 聚丙烯氨膠體(SDS-PAGE)電泳法……………………….23
4. 西方墨點法(western blot)…………………..………………23
5. 酵素結合免疫分析法(enzyme-linked immunosorbent assay；ELISA)……………………………………………..24
6. 過敏原檢測…………………………………………………24
7. 統計分析…………………………………………………25
肆. 實驗結果…………………………………………………………26
1. 研究對象之特徵……………………………………………26
2. 氣喘病人體內存在台灣常見過敏原IgE種類分析………27
3. ENO1蛋白的純化…………………………………………28
4. 利用西方墨點法確認ENO1蛋白…………………………29
5. 利用西方墨點法分析各組別血液當中anti-ENO1 抗體存在情形………………………………………………………29
6. 利用ELISA分析氣喘病人血液當中anti-ENO1 抗體存在情形………………………………………………………30
7. 利用ELISA分析高腫瘤標記血液當中anti-ENO1 抗體存在情形………………………………………………………30
伍. 討論………………………………………………………………32
1. 受試者資料分析……………………………………………32
2. 過敏原分析………………………………………………32
3. ENO1蛋白的純化…………………………………………34
4. ENO1蛋白的確認…………………………………………34
5. 氣喘病人ENO1抗體分析…………………………………35
6. 高腫瘤標記血清ENO1抗體分析…………………………36
7. Western bolt與ELISA結果不符合分析………………37
陸. 未來展望……………………………………………………………39
柒. 圖表…………………………………………………………………40
捌. 參考文獻……………………………………………………………52

1.Pancholi V, Multifunctional alpha-enolase: its role in diseases. Cell Mol Life, 2001. 58: p. 902-20.
2.Sabbatini A, D.M., Marchini B, Chimenti D, Moscato S, and e.a. Pratesi F, Alpha-enolase is a renal-specific antigen associated with kidney involvement in mixed cryoglobulinemia. Clin Exp Rheumatol, 1997. 15: p. 655-8.
3.Lopez-Alemany R, L.C., Hawley S, Mirshahi M, Fabregas P, Jardi M, Merton E, Miles LA & Felez J, Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-enolase. Am J Hematol, 2003. 72: p. 234-242.
4.Redlitz A, F.B., Plow EF, Miles LA, The role of an enolaserelated molecule in plasminogen binding to cells. Eur J Biochem, 1995. 227: p. 407-15.
5.Angiolella L, F.M., Stringaro A, Maras B, Simonetti N, Cassone A, Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J Infect Dis, 1996. 173(3): p. 689-90.
6.W, N., Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells. Traffic, 2005. 6: p. 607-14.
7.Seweryn E, P.J., Bednarz-Misa IS, Ceremuga I, Saczko J, Kulbacka J & Gamian A, Localization of enolase in the subfractions of a breast cancer cell line. Z Naturforsch C, 2009. 64: p. 754-58.
8.Saldanha RG, M.M., Bdeir K, Cines DB, Song X, Uitto PM, Weinreb PH, Violette SM & Baker MS, Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy. J Proteome Res, 2007. 6: p. 1016-28.
9.O, W., The Metabolism of Tumours. Constable, London., 1930.
10.Michela C, S.F., Paola C and Francesco N, alpha-enolase: a promising therapeutic and diagnostic tumor target. FEBS Journal, 2011. 28: p. 1064-74.
11.Zhou W, C.M., Fredolini C, Piemonti L, Liotta LA, Novelli F & Petricoin EF, Mass spectrometry analysis of the post-translational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells. J Proteome Res, 2010. 9: p. 2929-2936.
12.Tomaino B, C.P., Capello M, Fredolini C, Sperduti I, Migliorini P, Salacone P, Novarino A, Giacobino A, Ciuffreda L et al., Circulating autoantibodies to phosphorylated alpha-enolase are a hallmark of pancreatic cancer. J Proteome Res, 2011. 1: p. 105-112.
13.Tu SH, C.C., Chen CS, Tam KW, Wang YJ, Lee CH, Lin HW, Cheng TC, Huang CS, Chu JS, Increased expression of enolase alpha in human breast cancer confers tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat, 2010. 121: p. 539-53.
14.Shih NY, L.H., Chang GC, Lin HC, Wu YC, Liu JM, Liu KJ & Tseng SW, Anti-alpha-enolase autoantibodies are down-regulated in advanced cancer patients. Jpn J Clin Oncol, 2010. 40: p. 663-69.
15.Adrian PV, B., Oprins M, Rapola S, LahdenkariM, Kilpi T, et al., Development of antibodies against pneumococcal proteins alpha-enolase, immunoglobulin A1 protease, streptococcal lipoprotein rotamaseA, and putative proteinasematuration protein A in relation to pneumococcal carriage and otitis media. Vaccine, 2004. 22: p. 2737-42.
16.van Deventer AJ, v.V.H., Hop WC, Goessens WH., Diagnostic value of anti-Candida enolase antibodies. J Clin Microbiol, 1994. 32: p. 17-32.
17.Roozendaal C, Z.M., Horst G, Lockwood CM, Kleibeuker JH, Limburg PC., Catalase and alpha-enolase: two novel granulocyte autoantigens in inflammatory bowel disease (IBD). Clin Exp Immunol, 1998. 112: p. 10-6.
18.Orth T, K.R., Diekmann O, Faust J, Meyer zum Buschenfelde KH, Mayet WJ., Identification and characterization of autoantibodies against catalase and alpha-enolase in patients with primary sclerosing cholangitis. Clin Exp Immunol, 1998. 112: p. 507-17.
19.Lee KH, C.H., Kim HS, Oh SH, Ha MK, Baik JH, et al., Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behcet''s disease. Arthritis Rheum, 2003. 48: p. 2025-35.
20.Busse WW, B.-S.S., Wenzel SE., Pathophysiology of severe asthma. J Allergy Clin Immunuol, 2000. 106: p. 1033-42.
21.Lassalle P, D.Y., Gosset P, Gras-Masse H, Wallaert B, Tonnel AB., T and B cell immune response to a 55-kDa endothelial cell-derived antigen in severe asthma. Eur J Immunol, 1993. 23: p. 796-803.
22.E.D. Bateman, S.S.H., P.J. Barnes, J. Bousquet, M. FitzGerald, P. Gibson, K. Ohta, P. O’Byrne, S.E. Pedersen, E. Pizzichini, S.D. Sullivan, S.E. Wenzel and H.J. Zar, Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J, 2008. 31: p. 143-78.
23.楊明和、張翠霞, 中部某區域醫院以晶片法輔助小兒科醫師做過敏原檢測. 醫檢會報. 27(1): p. 24-31.
24.Yong, S.B., et al., Different profiles of allergen sensitization in different ages and geographic areas in Changhua, Taiwan. J Microbiol Immunol Infect, 2012.
25.DH Nahm, K.L., JY Shin,YM Ye,Y Kang and HS Park, Identification of a-enolase as an autoantigen associated with severe asthma. J ALLERGY CLIN IMMUNOL, 2006. 118: p. 376-381.
26.Spellberg BJ, F.S., Edwards Jr JE., Current treatment strategies for disseminated candidiasis. Clin Infect Dis, 2006. 42(2): p. 244-51.
27.Ejma M, M.-H.M., Gorczyca WA, Podemski R, Szymaniec S, Kuropatwa M, Rogozinska-Szczepka J & Bartnik W, Antibodies to 46-kDa retinal antigen in a patient with breast carcinoma and cancerassociated retinopathy. Breast Cancer Res Treat, 2008. 110: p. 269-271.