臺灣博碩士論文加值系統

背景:尿路上皮細胞癌是現行前十大癌症之一，尿路上皮細胞癌初期的症狀不明顯，其最主要的臨床發現為血尿，即使尿路上皮細胞癌復發率極高，若能於初期診斷，五年存活率將85%；近來在尿路上皮細胞癌的臨床研究，有兩大方向，一為在治療上，手術技術與化療標靶藥物的進展，另一研究重點乃在於生物標誌的探索，亦既如何以非侵入性的方法開發與建立尿路上皮細胞癌的診斷工具，併更進一步探討尿路上皮細胞癌的生物標誌之臨床應用。

因為尿路上皮細胞癌細胞與正常的泌尿道上皮細胞，均會分泌外吐小體至尿液中，而由先前分子生物學的研究顯示，外吐小體可攜帶尿路上皮細胞癌的訊息；因此，研究尿液外吐小體的蛋白質體或許更能尿路上皮細胞癌之與癌症有關蛋白質的變化，之前有些質譜研究，是以液相層析串聯質譜分析外吐小體；迄今，基質輔助雷射脫附游離飛行質譜(Matrix-assisted laser desorption/ionization time-of-flight ,MALDI-TOF)尚未被用來研究外吐小體的蛋白質體，基質輔助雷射脫附游離飛行質譜具備了樣品製備簡單與快速的優勢，本研究以基質輔助雷射脫附游離飛行質譜儀研究尿液外吐小體之蛋白質體。

實驗設計：從2012年到2015年，我們分析129位尿路上皮細胞癌患者和62位為對照組的病人之尿液外吐小體。利用基質輔助雷射脫附游離飛行質譜分析這些實驗組與對照組的尿液外吐小體之蛋白質萃取物。並用免疫組織化學染色（Immunohistochemical,IHC）分析另外一組122個尿路上皮細胞癌組織與26個正常組織，以驗證所發現的生物標誌物。

結果：由基質輔助雷射脫附游離飛行質譜圖中，有兩個訊號m / z 5593 (α-1抗胰蛋白酶的肽片段;診斷尿路上皮細胞癌之敏感性50.4％，特異性96.9％)與m / z 5947 (組織蛋白H2B1K之診斷尿路上皮細胞癌敏感性62.0％，特異性92.3％)可被用為診斷尿路上皮細胞癌的生物標誌物。 尿路上皮細胞癌患者，其尿液外吐小體若可檢測出組織蛋白H2B1K，會比尿路上皮細胞癌患者外吐小體無檢測出組織蛋白H2B1K者，有2.29倍復發與3.11倍癌症進展的風險。
免疫組織化學染色(IHC)驗證此二生物標誌物的結果發現，在尿路上皮細胞癌組織中，α1 - 抗胰蛋白酶和組織蛋白H2B1K的表現，顯著高於正常組織。而且，α1 - 抗胰蛋白酶和組織蛋白H2B1K與尿路上皮細胞癌的高度惡化與低度惡化有顯著關聯性。

結論：利用基質輔助雷射脫附游離飛行質譜分析尿液外吐小體，此二蛋白質α1 - 抗胰蛋白酶和組蛋白H2B1K的有無，可以幫助尿路上皮細胞癌的診斷和評估預後。

Purpose: Studies have focused on establishing noninvasive, rapid methods for discovering urothelial carcinoma (UC) biomarkers. The urinary exosome proteome is believed to directly reflect the proteome of UC, providing a suitable investigation resource. Increasingly applied matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry facilitates reliable clinical diagnosis of bacteria. MALDI-TOF spectrometry, a rapid analytical platform, has not been used for urinary exosome analysis. Therefore, we used it for determining UC biomarkers.
Experimental Design: From 2012 to 2015, we enrolled 129 consecutive patients with UC and 62 participants without UC. Exosomes from their urine were isolated, and the protein extracts of these exosomes were analyzed through MALDI-TOF spectrometry. Moreover, immunohistochemical (IHC) analysis of another 122 UC and 26 non-UC tissues was conducted to verify the discovered biomarkers.
Results: Two peaks at m/z 5593 (fragmented peptide of alpha-1-antitrypsin; sensitivity, 50.4%; specificity, 96.9%) and m/z 5947 (fragmented peptide of histone H2B1K sensitivity, 62.0%; specificity, 92.3%) were identified as UC diagnosis exosome biomarkers. UC patients with detectable histone H2B1K showed 2.29- and 3.11-fold increased risks of recurrence and progression, respectively, compared with those with nondetectable histone H2B1K. Verification results of IHC staining revealed significantly higher expression of alpha 1-antitrypsin (p = 0.038) and H2B1K (p = 0.005) in UC tissues than in normal tissues. The expression of alpha 1-antitrypsin and H2B1K in UC tissues was significantly correlated with UC grades (p < 0.05).
Conclusion: Urinary exosome proteins alpha 1-antitrypsin and histone H2B1K, which are identified through MALDI-TOF analysis, could facilitate rapid diagnosis and prognosis of UC.

中文摘要----------------------------------------------ii
英文摘要---------------------------------------------- iv
序---------------------------------------------------- vi
目錄-------------------------------------------------- vii
第一章 前言------------------------------------------- 1
第一節 研究背景------------------------------------ 1
第二節 研究目的------------------------------------ 4
第二章 研究方法--------------------------------------- 5
第一節 研究材料------------------------------------ 5
第二節 研究設計------------------------------------ 7
第三節 統計方法------------------------------------14
第三章 研究結果----------------------------------------15
第四章 討論-------------------------------------------22
第一節 結果討論------------------------------------22
第二節 其他相關性討論------------------------------26
第五章 結論與建議-------------------------------------31
第一節 結論----------------------------------------31
第二節 建議----------------------------------------31
第六章 圖與表格
圖一-----------------------------------------------32
圖二-----------------------------------------------33
表格一---------------------------------------------34
圖三-----------------------------------------------35
表格二---------------------------------------------36
圖四-----------------------------------------------37
圖五-----------------------------------------------38
補充表格一-----------------------------------------39
補充表格二-----------------------------------------40
補充圖一-------------------------------------------41
補充圖二-------------------------------------------42
補充圖三-------------------------------------------43
補充圖四-------------------------------------------44

第七章 參考文獻與附錄----------------------------------45

參考文獻
1Barratt, J.,Topham, P. Urine proteomics: the present and future of measuring urinary protein components in disease. Can Med Assoc J 177, 361-368 (2007).
2Thongboonkerd, V. Renal and Urinary Proteomics. Proteomics Clin Appl 2, 947-949 (2008).
3Quintana, L. F., Amanda Sole-Gonzalez, Susana G. Kalko, Elisenda Banon-Maneus, Manel Sole, Fritz Diekmann, et al. Urine proteomics to detect biomarkers for chronic allograft dysfunction. J Am Soc Nephrol 20, 428-435 (2009).
4Yamamoto, T., Langham, R. G., Ronco, P., Knepper, M. A., Thongboonkerd, V. Towards standard protocols and guidelines for urine proteomics: a report on the Human Kidney and Urine Proteome Project (HKUPP) Symposium and Workshop. Proteomics 8, 2156-2159 (2008).
5Kentsis A, Monigatti F, Dorff K, Campagne F, Bachur R, Steen H. Urine proteomics for profiling of human disease using high accuracy mass spectrometry. Proteomics Clin Appl 3, 1052-1061 (2009).
6Aebersold, R., Mann, M. Mass spectrometry-based proteomics. Nature 422, 198-207 (2003).
7Pisitkun, T., Shen, R.-F. & Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101, 13368-13373 (2004).
8Ploeg, M., Aben, K. K., Kiemeney, L. A. The present and future burden of urinary bladder cancer in the world. World J Urol 27, 289-293 (2009).
9Witjes JA, Comperat E, Cowan NC, De Santis M, Gakis G, Lebret T, et al. EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol 65, 778-792 (2014).
10Palou J, Rodriguez-Rubio F, Huguet J, Segarra J, Ribal MJ, Alcaraz A, et al. Multivariate analysis of clinical parameters of synchronous primary superficial bladder cancer and upper urinary tract tumor. J Urol 174, 859-861 (2005).
11Lotan, Y. & Roehrborn, C. G. Cost-effectiveness of a modified care protocol substituting bladder tumor markers for cystoscopy for the followup of patients with transitional cell carcinoma of the bladder: a decision analytical approach. J Urol 167, 75-79 (2002).
12Cheruvanky A, Zhou H, Pisitkun T, Kopp JB, Knepper MA, Yuen PS, et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292, 1657-1661 (2007).
13Duijvesz, D., Luider, T., Bangma, C. H.,Jenster, G. Exosomes as biomarker treasure chests for prostate cancer. Eur urol 59, 823-831 (2011).
14Kulasingam, V., Diamandis, E. P. Strategies for discovering novel cancer biomarkers through utilization of emerging technologies. Nat Rev Clin Oncol 5, 588-599 (2008).
15Oppenheim, J., Biragyn, A., Kwak, L.,Yang, D. Roles of antimicrobial peptides such as defensins in innate and adaptive immunity. Ann Rheum Dis 62, ii17-ii21 (2003).
16Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, et al. Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol 20, 363-379 (2009).
17M Hunt, J., Tuder, R. Alpha 1 anti-trypsin: one protein, many functions. Curr Mol Med 12, 827-835 (2012).
18Hosseini-Beheshti, E., Pham, S., Adomat, H., Li, N., Guns, E. S. Exosomes as biomarker enriched microvesicles: characterization of exosomal proteins derived from a panel of prostate cell lines with distinct AR phenotypes. Mol Cell Proteomics 11, 863-885 (2012).
19Yang P, Sun Z, Krowka MJ, Aubry MC, Bamlet WR, Wampfler JA , et al. Alpha1-antitrypsin deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer risk. Arch Intern Med 168, 1097-1103 (2008).
20Kuvibidila, S., Rayford, W. Correlation between serum prostate-specific antigen and alpha-1-antitrypsin in men without and with prostate cancer. J Lab Clin Med 147, 174-181 (2006).
21Rajendiran S, Parwani AV, Hare RJ, Dasgupta S, Roby RK, Vishwanatha JK. MicroRNA-940 suppresses prostate cancer migration and invasion by regulating MIEN1. Mol Cancer 13, 1 epup (2014).
22Sylvester RJ, van der Meijden AP, Oosterlinck W, Witjes JA, Bouffioux C, Denis L, N, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. Eur Urol 49, 466-477 (2006).
23Clague, M. J., Coulson, J. M., Urbe, S. Deciphering histone 2A deubiquitination. Genome Biol. 9, 1 epup (2008).
24Cole, A. J., Clifton-Bligh, R., Marsh, D. J. Histone H2B monoubiquitination: roles to play in human malignancy. Endocr Relat Cancer 22, T19-T33 (2015).
25Kondo, Y. , Issa, J.-P. J. Epigenetic changes in colorectal cancer. Cancer Metastasis Rev 23, 29-39 (2004).
26D''Arcy, P., Wang, X., Linder, S. Deubiquitinase inhibition as a cancer therapeutic strategy. Pharmacol Ther 147, 32-54 (2015).
27Telu KH1, Abbaoui B, Thomas-Ahner JM, Zynger DL, Clinton SK, Freitas MA, et al. Alterations of histone H1 phosphorylation during bladder carcinogenesis. J Proteome Res 12, 3317-3326 (2013).
28Loddo M1, Kingsbury SR, Rashid M, Proctor I, Holt C, Young J, et al. Cell-cycle-phase progression analysis identifies unique phenotypes of major prognostic and predictive significance in breast cancer. Br J Cancer 100, 959-970 (2009).
29Bonenfant, D. et al. Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry. Mol Cell Proteomics 6, 1917-1932, (2007).
30Fromont G, Roupret M, Amira N, Sibony M, Vallancien G, Validire P, et al. Tissue microarray analysis of the prognostic value of E-cadherin, Ki67, p53, p27, survivin and MSH2 expression in upper urinary tract transitional cell carcinoma. Eur Urol 48, 764-770 (2005).
31Nakanishi K1, Hiroi S, Tominaga S, Aida S, Kasamatsu H, Matsuyama S, et al. Expression of hypoxia-inducible factor-1alpha protein predicts survival in patients with transitional cell carcinoma of the upper urinary tract. Clin Cancer Res 11, 2583-2590 (2005).
32Shariat SF, Karakiewicz PI, Godoy G, Karam JA, Ashfaq R, Fradet Y, et al. Survivin as a prognostic marker for urothelial carcinoma of the bladder: a multicenter external validation study. Clin Cancer Res 15, 7012-7019 (2009).
33Saito K, Kawakami S, Ohtsuka Y, Fujii Y, Masuda H, Kumagai J, et al. The impact of preoperative serum C-reactive protein on the prognosis of patients with upper urinary tract urothelial carcinoma treated surgically. BJU international 100, 269-273, (2007).
34Lotan Y, Bagrodia A, Passoni N, Rachakonda V, Kapur P, Arriaga Y, et al. Prospective evaluation of a molecular marker panel for prediction of recurrence and cancer-specific survival after radical cystectomy. Eur Urol 64, 465-471, (2013).
35Zhang, Y., Fonslow, B. R., Shan, B., Baek, M. C., Yates, J. R. Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113, 2343-2394 (2013).
36Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., Whitehouse, C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64-71 (1989).
37Wilm, M. Principles of electrospray ionization. Mol Cell Proteomics 10, M111. 009407 (2011).
38Tang, L., Kebarle, P. Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution. Anal Chem 65, 3654-3668 (1993).
39Wilm, M., Mann, M. Analytical properties of the nanoelectrospray ion source. Anal Chem 68, 1-8 (1996).
40Juraschek, R., Dulcks, T. & Karas, M. Nanoelectrospray—more than just a minimized-flow electrospray ionization source. J Am Soc Mass Spectrom 10, 300-308 (1999).
41Karas, M. & Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60, 2299-2301 (1988).
42Chen CJ, Lai CC, Tseng MC, Liu YC, Lin SY, Tsai FJ. et al. Simple fabrication of hydrophobic surface target for increased sensitivity and homogeneity in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of peptides, phosphopeptides, carbohydrates and proteins. Anal Chim Acta 783, 31-38 (2013).
43Bonk, T., Humeny, A. MALDI-TOF-MS analysis of protein and DNA. Neuroscientist 7, 6-12 (2001).
44Bucknall, M., Fung, K. Y., Duncan, M. W. Practical quantitative biomedical applications of MALDI-TOF mass spectrometry. J Am Soc Mass Spectrom 13, 1015-1027 (2002).
45Yates, J. R., Cociorva, D., Liao, L., Zabrouskov, V. Performance of a linear ion trap-Orbitrap hybrid for peptide analysis. Anal Chem 78, 493-500 (2006).
46Mamyrin, B. Time-of-flight mass spectrometry (concepts, achievements, and prospects). Int J Anal Mass Spectrom 206, 251-266 (2001).
47Doroshenko, V. M., Cotter, R. J. Ideal velocity focusing in a reflectron time-of-flight mass spectrometer. J Am Soc Mass Spectrom 10, 992-999 (1999).
48Lu, J. J., Tsai, F. J., Ho, C. M., Liu, Y. C., Chen, C. J. Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF. Anal Chem 84, 5685-5692 (2012).
49Tang, N., Tornatore, P., Weinberger, S. R. Current developments in SELDI affinity technology. Mass Spectrom Rev 23, 34-44 (2004).
50Albrethsen, J. Reproducibility in protein profiling by MALDI-TOF mass spectrometry. Clin Chem 53, 852-858 (2007).
51Ye, H., Sun, L., Huang, X., Zhang, P., Zhao, X. A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ labeling and SCX-LC-MS/MS. Mol Cell Biochem 343, 91-99 (2010).