臺灣博碩士論文加值系統

食道癌依其病理型態主要可分為麟狀上皮細胞癌 (squamous cell carcinoma) 和腺癌 (adenocarcinoma) 兩種，臨床上的比例約為8：2。目前對於評估食道癌的預後以TNM分級及pathological pN (node) 的狀態為預測因子。TNM系統是食道癌分級最常用的系統，T代表腫瘤大小和它散佈的程度，N代表侵襲到淋巴結的程度，M為轉移至其他器官的狀況；而依腫瘤侵襲的深度分為四等級：包括表皮層 (epithelium, EP)、黏膜間質層 (lamina propria mucosa, LPM)、黏膜肌層 (muscularis mucosa, MM) 和黏膜下層 (submucosa, SM) 。癌症的分期是用來評估癌細胞的散佈和侵襲至多遠位置的指標，這會決定治療的方式。
食道癌是非常惡性的腫瘤，因為其早期症狀不明顯；在表淺性食道癌病患，只有12.1％會出現吞厭不舒服及疼痛的現象。當出現吞厭困難、體重快速減輕、胸痛、吞厭疼痛沙啞等症狀時，一般只有40％病患可以接受外科手術切除，但是併發症很高，因此其五年存活率較其他癌症相對為低. 癌症的分期是用來評估癌細胞的散佈和侵襲至多遠位置的指標，這會決定治療的方式。雖然TNM tumor staging與接受curative esophagectomy之後的臨床預後具有關聯性，但卻與癌細胞分化程度及對淋巴血管系統之侵襲能力的相關性不高 (Wang et al., 1999)，因此僅採用TNM tumor staging 用來評估病患疾病的整體進展及預後並不足夠 (Sobin, 2003)；因此，本研究期望藉由發現novel gene expression當做新的生物指標用以評估國人食道癌此疾病的臨床進展。
而在本篇論文中，我們利用蛋白質體技術去分析利用in vitro Invasion Assay所篩選的親代的食道癌細胞(ECA為親代細胞株)和第一代培養較惡性的食道癌細胞(ECA1-1為第一次篩選之下層細胞株)及 ECA1-4(為第四次篩選之下層細胞株)所表達蛋白質的差異，並將這些有差異的蛋白質利用質譜儀鑑定出來。並用RT-PCR和西方點墨法(Western blot)分析特異性蛋白質的表現，進而探討這基因在食道癌所扮演的角色為何。

The purpose of this thesis is to identify the potential novel gene from one Taiwanese esophageal cancer cell line(CE 81T/VGH) this is research except to find novel gene expression for
new biomarker which to estimate patients’progression and whole progress. In this research ,we use proteomic tool,such as 2D electrophoresis to analyze in vitro Invasion Assay which select parent cell(ECA), one generation malignant (ECA1-1), forth generation malignant (ECA1-4) to find protein difference in Mass/Mass identify, anduse Western blot and RT-PCR to analyze protein expression, andthan discuss this gene charter in esophagus cancer.The cancer stage is used to estimate the cancer cell spread and metastasis.This is will decide the cure manner. Although TNM tumor stagingand curative esophagectomy which has relation for progression,less relation for cancer cell division and lymph node metastasis. It is not enough used TNM tumor staging toestimate patients’ progression and whole progress. Sothis is research except to find novel gene expression fornew biomarker which to estimate patients’progression and whole progress.

中文摘要 -------------------------------------------- 2
英文摘要 -------------------------------------------- 4
縮寫 -------------------------------------------- 6
前言 -------------------------------------------- 7
實驗材料 -------------------------------------------- 13
實驗方法 -------------------------------------------- 17
結果 -------------------------------------------- 35
圖表 -------------------------------------------- 37
文獻回顧 -------------------------------------------- 48
討論 -------------------------------------------- 51
參考文獻 -------------------------------------------- 56

Chun-Xia Yang, Keitaro Matsuo, Zhi-Ming Wang, Kazuo Tajima. Phase I/II enzyme gene polymorphisms and esophageal cancer risk: A meta-analysis of the literature World J Gastroenterol;11(17):2531-2538(2005)
Gen Tamura. Alterations of tumor suppressor and tumor-related genes in the development and progression of gastric cancer. World J Gastroenterol January 14; 12(2):192-198(2006)
Gevaert K, Vandekerckhove J. 2000. Protein identification methods in proteomics. Electrophoresis 21:1145-1154. Jan Brabender, M.D., Reginald V. Lord, M.B.B.S., Kumari Wickramasinghe, M.D., Ralf Metzger, M.D., Paul M. Schneider, M.D., Ji-Min Park, M.S., Arnulf H. Holscher, M.D., Tom R. DeMeester, M.D., Kathleen D. Danenberg, B.S., Peter V. Danenberg, Ph.D. Glutathione S–Transferase-Pi Expression Is Downregulated in Patients With Barrett’s Esophagus and Esophageal Adenocarcinoma. Journal of Gastrointestinal Surgery Vol. 6, No. 3 2002
Jin-Tang Dong. Prevalent Mutations in Prostate Cancer. Journal of Cellular Biochemistry .97:433–447 (2006)
Masaru Shinozaki, Dave S.B. Hoon, Armando E. Giuliano, Nora M. Hansen, He-Jing Wang, Roderick Turner, and Bret Taback1. Distinct Hypermethylation Profile of Primary Breast Cancer Is Associated with Sentinel Lymph Node Metastasis. Clinical Cancer Research .Vol. 11, 2156–2162, March 15, (2005)
Mary-Beth Moore Joshi, Yoshinori Shirota,Kathleen D. Danenberg, Debbi H. Conlon, Dennis S. Salonga, James E. Herndon II, Peter V. Danenberg, and David H. Harpole, Jr. High Gene Expression of TS1, GSTP1, and ERCC1 Are Risk Factors for Survival in Patients Treated with Trimodality Therapy for Esophageal Cancer. Clinical Cancer Research Vol. 11, 2215–2221, March 15, 2005
Masashi Nakayama, Mark L. Gonzalgo, Srinivasan Yegnasubramanian, Xiaohui Lin,Angelo M. De Marzo, and William G. Nelson1. GSTP1 CpG Island Hypermethylation as a Molecular Biomarker for Prostate Cancer Journal of Cellular Biochemistry. 91:540–552 (2004)
Jan Brabender, M.D., Reginald V. Lord, M.B.B.S., Kumari Wickramasinghe, M.D.,
Ralf Metzger, M.D., Paul M. Schneider, M.D., Ji-Min Park, M.S., Arnulf H. Holscher, M.D.,Tom R. DeMeester, M.D., Kathleen D. Danenberg, B.S., Peter V. Danenberg, Ph.D. Glutathione S–Transferase-Pi Expression Is Downregulated in Patients With Barrett’s Esophagus and Esophageal Adenocarcinoma. Journal of Gastrointestinal Surgery. Vol. 6, No. 3 2002
McDonald WH, Yates JR, 3rd. 2000. Proteomic tools for cell biology. Traffic 1:747-754.
Pandey A, Mann M. 2000. Proteomics to study genes and genomes. Nature 405:837-846.
Peng J, Gygi SP. 2001. Proteomics: the move to mixtures. J Mass Spectrom 36:1083-1091.
Queeny K.Y. Chan,Ui-Soon Khoo,1HextanY.S. Ngan, Chong-QingYang,Wei-Cheng Xue,1 KelvinY.K. Chan, Pui-Man Chiu,Philip P.C. Ip,and Annie N.Y. Cheung. Single Nucleotide Polymorphismof Pi-Class Glutathione S-Transferase and Susceptibility to Endometrial Carcinoma .Clin Cancer Res 2981 2005;11(8) April 15, 2005
Quadroni M, James P. 1999. Proteomics and automation. Electrophoresis 20:664-677.
Riichiroh Maruyama, Shinichi Toyooka, Kiyomi O. Toyooka, Kenichi Harada, Arvind K. Virmani, Sabine Zo¨chbauer-Mu¨ ller, Alfredo J. Farinas, Funda Vakar-Lopez, John D. Minna, Arthur Sagalowsky, Bogdan Czerniak, and Adi F. Gazdar. Aberrant Promoter Methylation Profile of Bladder Cancer and Its Relationship to Clinicopathological Features .CANCER RESEARCH 61, 8659–8663, December 15, (2001)
Rabilloud T. 2000. Detecting proteins separated by 2-D gel electrophoresis. Anal Chem 72:48A-55A.8:181-190.
S. Aliya, P. Reddanna and K. Thyagaraju. Does glutathione S-transferase Pi (GST-Pi) a marker protein for cancer? Molecular and Cellular Biochemistry 253: 319–327, 2003.
Sherr CJ, Roberts JM. 1995. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149-1163.
Shevchenko A, Wilm M, Vorm O, Mann M. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850-858.
Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams KL. 1996. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13:19-50.
Yutaka Tokumaru, Susan V. Harden, Dong-Il Sun,Keishi Yamashita, Jonathan I. Epstein,and David Sidransky. Optimal Use of a Panel of Methylation Markers with GSTP1 Hypermethylation in the Diagnosis of Prostate Adenocarcinoma Clinical. Cancer Research Vol. 10, 5518–5522, August 15( 2004)
黃彥博. 2004. 鼠骨骼肌肉細胞株C2C12進行肌肉分化之蛋白質體研究.
李君豐. 2005. 老鼠骨骼肌肉細胞株C2C12之肌肉分化因子研究
Benford, D., Boyle, C., Dekant,W., Fuchs, R., Gaylor, D.W., Hard, G.,McGregor, D.B., Pitt, J.I., Plestina, R., Shephard, G., Solfrizzo, M. Verger, P.J.P., Walker, R., 2001. Ochratoxin A. In: Safety Evaluationsof Certain Mycotoxins in Food, WHO Food Additives Series47, FAO Food and Nutrition Paper 74, IPCS International Programmeon Chemical Safety. WHO, Geneva, pp. 281–387.
Castegnaro, M., Chernozemsky, I.N., Hietanen, E., Bartsch, H., 1990. Are mycotoxins risk factors for endemic nephropathy andassociated urothelial cancers? Arch. Geschwulstforsch. 60, 295–303.
Chernozemsky, I.N., 1991. Balkan Endemic Nephropathy and theAssociated Tumours of the Urinary System: a Summary of EpidemiologicalFeatures in Bulgaria, vol. 115. IARC Sci. Publ, pp.3–4.
Dai, J., Park, G., Wright,M.W., Adams, M., Akman, S.A., Manderville,R.A., 2002. Detection and characterization of a glutathione conjugateof ochratoxin A. Chem. Res. Toxicol. 15, 1581–1588.
De Groene, E.M., Jahn, A., Horbach, G.J., Fink-Gremmels, J., 1996.Mutagenicity and genotoxicity of the mycotoxin ochratoxin A.Environ. Toxicol. Pharmacol. 1, 21–26.
Deutsche Forschungsgemeinschaft (DFG), 2003. List of MAK and
BAT Values, Commission for the Investigation of Health Hazards
of Chemical Compounds in the Work Area, Report No. 39.
Wiley–VCH, Verlag Weinheim.Di Paolo, N., Guarnieri, A., Garosi, G., Sacchi, G., Mangiarotti, A.M.,Di Paolo, M., 1994. Inhaled mycotoxins lead to acute renal failure.Nephrol. Dial. Transplant. 9 (Suppl. 4), 116–120.
Domijan, A.-M., Zeljezic, D., Kopjar, N., Paraica, M., 2006. Standard
and Fpg-modified comet assay in kidney cells of ochratoxin A- and
fumonisin B1-treated rats. Toxicology 222, 53–59.
D¨orrenhaus, A., Flieger, A., Golka, K., Schulze, H., Albrecht, M.,
Degen, G.H., F¨ollmann,W., 2000. Induction of unscheduled DNA
synthesis in primary human urothelial cells by the mycotoxin
ochratoxin A. Toxicol. Sci. 53, 271–277.
El Adlouni, C., Pinelli, E., Azemar, B., Zaoui, D., Beaune, P., Pfohl-Leszkowicz, A., 2000. Phenobarbital increases DNA adduct andmetabolites formed by ochratoxin A: role of CYP 2C9 andmicrosomal glutathione S-transferase. Environ. Mol. Mutagen. 35,123–131.
Flieger, A., D¨orrenhaus, A., Golka, K., Schulze, H., F¨ollmann, W.,1998. Genotoxic effect of the mycotoxin ochratoxin A in culturedhuman urothelial cells. Occupational Hyg. 4, 297–307.
Garte, S., Gaspari, L., Alexandrie, A.K., Ambrosone, C., Autrup, H.,Autrup, J.L., Baranova, H., Bathum, L., Benhamou, S., Boffetta, P.,Bouchardy, C., Breskvar, K., Brockmoller, J., Cascorbi, I., Clapper,M.L., Coutelle, C., Daly, A., Dell’Omo, M., Dolzan, V., Dresler,C.M., Fryer, A., Haugen, A., Hein, D.W., Hildesheim, A., Hirvonen,A., Hsieh, L.L., Ingelman-Sundberg, M., Kalina, I., Kang,
D., Kihara, M., Kiyohara, C., Kremers, P., Lazarus, P., Le Marchand,
L., Lechner, M.C., van Lieshout, E.M., London, S., Manni,J.J., Maugard, C.M., Morita, S., Nazar-Stewart, V., Noda, K., Oda,Y., Parl, F.F., Pastorelli, R., Persson, I., Peters, W.H., Rannug, A.,Rebbeck, T., Risch, A., Roelandt, L., Romkes, M., Ryberg, D.,Salagovic, J., Schoket, B., Seidegard, J., Shields, P.G., Sim, E., Sinnet,
D., Strange, R.C., Stucker, I., Sugimura, H., To-Figueras, J.,Vineis, P., Yu, M.C., Taioli E, 2001. Metabolic gene polymorphismfrequencies in control populations. Cancer Epidemiol. BiomarkersPrev. 10, 1239–1248.
Gautier, J., Richoz, J., Welti, D.H., Markovic, J., Gremaud, E., Guengerich,
F.P., Turesky, R.J., 2001. Metabolism of ochratoxin A:
zymes. Chem. Res. Toxicol. 14, 34–45.
Gillman, I.G., Clark, T.N., Manderville, R.A., 1999. Oxidation ofochratoxin A by an Fe-porphyrin system: model for enzymaticactivation andDNAcleavage. Chem. Res. Toxicol. 12, 1066–1076.
Grosse, Y., Monje, M.C., Mac´e, K., Pfeifer, A., Pfohl-Leszkowicz,A., 1997. Use of bronchial epithelial cells expressing humancytochrom P450 for study on metabolism and genotoxicity ofochratoxin A. In vitro Toxicol. 10, 93–102.
Gross-Steinmeyer, K., Weymann, J., Hege, H.G., Metzler, M., 2002.Metabolism and lack of DNA reactivity of the mycotoxin ochratoxina in cultured rat and human primary hepatocytes. J. Agric.Food Chem. 50, 938–945.
Hurst, R., Bao, Y., Jemth, P., Mannervik, B., Williamson, G., 1998.Phospholipid hydroperoxide glutathione peroxidase activity ofhuman glutathione transferases. Biochem. J. 332, 97–100.
International Agency for Research on Cancer (IARC), 1993. OchratoxinA. In: IARC Monographs on the Evaluation of the CarcinogenicRisks to Humans: Some Naturally Occurring Substances.Food Items and Constituents, Heterocyclic Aromatic Amines andMycotoxins, vol. 56. International Agency for Research on Cancer,Geneva, Lyon, pp. 26–32.
Kempkes, M., Golka, K., Reich, S., Reckwitz, T., Bolt, H.M., 1996.Glutathione S-transferase GSTM1 and GSTT1 null genotypes aspotential risk factors for urothelial cancer of the bladder. Arch.Toxicol. 71, 123–126.
Ko, Y., Koch, B., Harth, V., Sachinidis, A., Thier, R., Vetter, H., Bolt,H.M., Br¨uning, T., 2000. Rapid analysis of GSTM1, GSTT1 andGSTP1 polymorphisms using real-time polymerase chain reaction.Pharmacogenetics 10, 271–274.
Kuramochi, G., Gekle, M., Silbernagl, S., 1997a. Derangement of pHhomeostasis in the renal papilla: ochratoxin A increases pH in vasarecta blood. Nephron 76, 472–476.
Kuramochi, G., Gekle, M., Silbernagl, S., 1997b. Ochratoxin A disturbspH homeostasis in the kidney: increases in pH and HCO3−in the tubules and vasa recta. Pfl¨ugers Arch. 434, 392–397.
Landi, S., Norppa, H., Frenzilli, G., Cipollini, G., Ponzanelli, I., Barale,R., Hirvonen, A., 1998. Individual sensitivity to cytogeneticeffects of 1,2,3,4-diepoxybutane in cultured human lymphocytes:influence of glutathione S-transferase M1, P1 and T1 genotypes.Pharmacogenetics 8, 461–471.
Lebrun, S., F¨ollmann, W., 2002. Detection of ochratoxin A-inducedDNA damage in MDCK cells by alkaline single cell gel electrophoresis(comet assay). Arch. Toxicol. 75, 734–741.
Le Tutour, B., Tantaoui-Elaraki, A., Ihlal, L., 1983. Simultaneousdetection of aflatoxin B1 and ochratoxin A in olive oil. J. Am.Oil Chem. Soc. 60, 835–837.
Lewis, C.W., Smith, J.E., Anderson, J.G., Freshney, R.I., 1999.Increased cytotoxicity of food-borne mycotoxins toward humancell lines in vitro via enhanced cytochrome p450 expressionusing the MTT bioassay. Mycopathologia 148, 97–102.
Malaveille, C., Brun, G., Bartsch, H., 1994. Structure–activity studiesin E. coli strains on ochratoxinA(OTA) and its analogues implicatea genotoxic free radical and acytotoxic thiol derivative as reactivemetabolites. Mutat. Res. 307, 141–147.
Manderville, R.A., 2005. A case for the genotoxicity of ochratoxin A
by bioactivation and covalentDNAadduction. Chem. Res. Toxicol.
18, 1091–1097.
Meki, A.R., Hussein, A.A., 2001. Melatonin reduces oxidative stress
induced by ochratoxin A in rat liver and kidney. Comp. Biochem.Physiol. C Toxicol. Pharmacol. 130, 305–313.
O‘Brien, E., Dietrich, D.R., 2005. Ochratoxin A: the continuingenigma. Crit. Rev. Toxicol. 35, 33–60.
Payen, J., Girad, T., Gaillardin, M., Lafont, P., 1983. About contamination
of beers by mycotoxins. Microbiol. Alim. Nutr. 1, 143–146.
Petkova-Bocharova, T., Castegnaro, M., 1991. Ochratoxin A in human
blood in relation to Balkan endemic nephropathy and urinary tract
tumors in Bulgaria. In: Castegnaro, M., Plestina, R., Dierheimer,
G., Chernozemsky, I.N., Bartsch, H. (Eds.), Mycotoxins, Endemic
Nephropathy and Urinary Tract Tumors, vol. 115. IARC Sci. Publ.,
pp. 135–137.
Pfohl-Leszkowicz, A., Pinelli, E., Bartsch, H., Mohr, U., Castegnaro,M., 1998. Sex- and strain-specific expression of cytochrom P450sin ochratoxin A-induced genotoxicity and carcinogenicity in rats.Mol. Carcinog. 23, 76–85.
Ringot, D., Chango, A., Schneider, Y.-J., Larondelle, Y., 2006. Toxicokinetics
and toxicodynamics of ochratoxin A, an update. Chem.
Biol. Interact. 159, 18–46.
Rosner, H., Bresch, H., Cholmakow-Bodechtel, C., Engel, G., Gareis,
M., Majerus, P., Wolff, J., Peiker, G., Rohrmann, B., 1998.
Ochratoxin-A-Gehalt in Serumproben der deutschen Bev¨olkerung.
Infektionsepidemiologische Forschung II 98, 11–12.
Schaaf, G.J., Nijmeijer, S.M., Maas, R.F., Roestenberg, P., de Groene,
E.M., Fink-Gremmels, J., 2002. The role of oxidative stress in the
ochratoxin A-mediated toxicity in proximal tubular cells. Biochim.
Biophys. Acta 1588, 149–158.
Scheuer, R., Leistner, L., 1985. Occurence of ochratoxin A in pork and
pork products. In: Proceedings of the European Meeting of MeatRes. Workers no. 32, vol. 1, 4:2, p. 191.
Schweighardt, H., Leibetseder, J., 1981. Nachweis von Mycotoxinen
mittels HPLC. Wiener Tier¨arztl. Wochenschr. 68, 302–305.
Simarro Doorten, A.Y., Bull, S., van der Doelen, M.A., Fink-
Gremmels, J., 2004. Metabolosm-mediated cytotoxicity of ochratoxin
A. Toxicol. in Vitro 18, 271–277.
Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple
echnique for quantitation of low levels of DNA damage inindividual cells. Exp. Cell Res. 175, 184–191.
Sostaric, B., Vukelic, M., 1991. Characteristics of urinary tract tumors in the area of Balkan endemic nephropathy in Croatia.In: Castegnaro, M., Plestina, R., Dierheimer, G., Chernozemsky,I.N., Bartsch, H. (Eds.), Mycotoxins, Endemic Nephropathyand Urinary Tract Tumors, vol. 115. IARC Scientific Publications/International Agency for Research on Cancer, Lyon, pp.29–35.
Steinhoff, C., Franke, K.H., Golka, K., Thier, R., R¨omer, H.C., R¨otzel,
C., Ackermann, R., Schulz, W.A., 2000. Glutathione transferase
isoenzyme genotypes in patients with prostate and bladder carcinoma.
Arch. Toxicol. 74, 521–526.
Stoev, S.D., 1998. The role of ochratoxin A as a possible cause of
Balkan endemic nephropathy and its risk evaluation. Vet. Hum.
Toxicol. 40, 352–360.
Sweeney, C., Farrow, D.C., Schwartz, S.M., Eaton, D.L., Checkoway,
H., Vaughan, T.L., 2000. Glutathione S-transferase M1, T1, and
P1 polymorphisms as risk factors for renal cell carcinoma: a casecontrol
study. Cancer Epidemiol. Biomarkers Prev. 9, 449–454.
Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A.,Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F.,2000. Single cell gel/comet assay: guidelines for in vitro and in vivogenetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221.
Turesky, R.J., 2005. Perspective: ochratoxin A is not a genotoxic carcinogen.
Chem. Res. Toxicol. 18, 1082–1090.
Wood, G.M., Patel, S., Entwisle, A.C., Williams, A.C., Boenke, A.,
Farnell, P.J., 1997. Ochratoxin A in wheat: certification of two
reference materials. Food Addit. Contam. 14, 237–248.
Zepnik, H., Pahler, A., Schauer, U., Dekant, W., 2001. Ochratoxin Ainduced
tumor formation: is there a role of reactive ochratoxin A
metabolites? Toxicol. Sci. 59, 59–67.