Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis.

Abstract

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.

Full text

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南台科技大學
生物科技研究所
碩士學位論文
利用蛋白質體學技術尋找與台灣地區移
形細胞惡性腫瘤相關的蛋白質
Search for the tumor-related proteins of
transition cell carcinoma in Taiwan by
proteomic analysis
研究生：姚雲欽
指導教授：吳定峰 博士
中華民國九十四年七月

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中文摘要
膀胱癌是台灣泌尿系統中常見的癌症之一，而移形細胞惡性腫瘤（TCC）是
膀胱癌中最常見一種。為了了解細胞惡性腫瘤致癌過程，我們利用蛋白質體學的方
法來建立移形細胞惡性腫瘤的蛋白質圖譜，在利用二維電泳與質譜儀的比較 grade
III對 grade II 的圖譜後，鑑定出七個在 grade III TCC 中表現下降的蛋白質和三個在
grade III TCC中表現上升的蛋白質，而這些表現差異中的 fatty acid binding proteins,
annexin V, heat shock protein 27和 lactate dehydrogenas chain H 在過去研究中已經被
初步證實與膀胱癌有相關聯，在本論文中發現到過去文獻中尚未發現的蛋白質，包
括有 annexin I, galectin-1, lysophospholipase 以及 mitochondrial short-chain
enoyl-coenzyme A hydratase 1 precursor，這些結果說明在不同癌化層級間，蛋白質表
現量的變化可能作為膀胱癌的分子符號，另外，此研究的成果可提供往後在泌尿系
統癌化初期病理機制更深入研究的基礎。
Annexin I 抗體的免疫組織化學染色實驗，證明 annexin I蛋白質在 Grade III 中
是呈現正向調節的結果與二維電泳是一致的。

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Abstract
Bladder cancer is one of the most prevalent cancers in urinary tract in Taiwan. To
better understand the carcinogenesis of bladder, we utilized proteomic approach to
establish the protein map and search for potential biomarkers of transitional cell
carcinoma (TCC), the major type of bladder cancer. Analysis by two-dimensional (2-D)
gel electrophoresis and tandem mass spectrometry showed that seven proteins were
down-regulated and three proteins were up-regulated in Grade III samples as compared
with those of Grade II. Of these down-regulated proteins, fatty acid binding proteins,
annexin V, heat shock protein 27, and lactate dehydrogenase have been shown to be
associated with bladder cancer. Our studies also found altered expression of a group of
proteins that have not been documented previously in bladder cancer, including annexin I,
galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A
hydratase 1 precursor. These results illustrated a pattern of differential protein expression
between low and high Grade tumors and it may be utilized as the molecular
fingerprinting of a subset of bladder cancers. In addition, this study provides a valuable
foundation to study pathological mechanisms in cancers of urothelial origin.
Consistent with the results of 2-D gels, the immunohistochemical staining of grade III
TCC with antiserum to annexin I protein demonstrated that the annexin I protein is
up-regulated in grade III TCC.

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致謝
兩年的時間很快就過去了，這段時間內承蒙吳定峰老師的教導，不時給予我在
學術實驗觀念上指正，更在論文上給予發揮空間，讓我在研究領域中收穫良多，增
加了許多興趣及信心，也謝謝成大微免所劉校生老師以及中正大學陳浩仁老師百忙
之中在實驗邏輯和論文寫作上的叮嚀校正，使論文更趨於完整，在此也獻上最大的
謝意。

在整個學習的過程中，感謝奇美泌尿科沈坤宏醫師在實驗材料上的補充供應，
成大蔣榮先老師生物資訊軟體的協助，吳烘老師不嗇的教導才能使本實驗順遂，也
感謝奇美張菁萍老師以及學長姐致良、瓊月、奇華、雲山、瑞菁在實驗和生活上的
幫忙，感謝實驗室合真、美員、侑遜、怡君、園堤、怡蜓在課業及休閒上的參與和
陪伴，使得求學生活更加充實，謝謝你們。

最後感謝我的家人在生活上的肯定與鼓勵，以及孟奇在精神上的支持與包容，
讓我在兩年中的生活沒有後顧之憂，也由衷的感激。

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目錄

第一章 前言 ...........................................................................................................1
第二章 文獻探討 …………………………………………………………………. 2
2-1. 膀胱癌的流行病學 …………………………………………………………
2
2-2. 膀胱癌的致病原因…………………………………………………………..
2
2-3. 膀胱癌的病理種類 …………………………………………………………
4
2-4. 膀胱癌的臨床病徵 ………………………………………………………… 6
2-5. 膀胱癌的診 ………………………………………………………………… 6
2-6. 膀胱癌的病理分期 ………………………………………………………… 8
2-7. 膀胱癌的治療 ……………………………………………………………… 8
2-8. 膀胱癌的預後 ……………………………………………………………… 9
2-9. 蛋白質體學 ………………………………………………………………… 10
2-9-1. 蛋白質二維電泳 …………………………………………………………. 11
2-9-2. 蛋白質鑑定技術 …………………………………………………………. 11
2-10. 研究膀胱癌的目的 ...................................................................................... 12
第三章 材料與方法 …………………………………………………………………
13
3-1. 材料與藥品 ……………………………………………………………….. 13
3-1-1. 人類移形性上皮細胞癌組織 ……………………………………………. 13
3-1-2. 藥品 ………………………………………………………………………. 13
3-1-3. 實驗儀器 …………………………………………………………………. 14
3-2. 研究方法 …………………………………………………………………….15
3-2-1. 移行性上皮細胞癌蛋白質萃取液之製備 ………………………………. 15
3-2-2. 蛋白質液定量 ……………………………………………………………. 15
3-2-3. 蛋白質等電點聚焦電泳法(isoelectric focusing, IEF) ………………….. 16
3-2-4. 膠條平衡（Equilibration） ………………………………………………. 16
3-2-5. SDS-聚丙烯膠電泳（SDS-polyacrylamide gel electrophoresis） ………..16