中文摘要
大腸癌的發生通常是漸進式的，而且包含了許多基因的突變，正常的大腸粘膜上皮細胞會因突變而過度增生，轉成為早期腺瘤，繼而生成惡性瘤繼而再轉移至身體其他器官。大腸癌致病機轉中牽涉到許多的基因及蛋白質。在基因階層，包括有APC基因、p53基因、 k-ras基因以及microsattilite instability等。其中，非遺傳性突變的APC基因發生突變密集區是在胺基酸序列1286~1513之間；p53基因發生點突變的位置分散於整個基因，並有3個突變熱點： 175、248、273及附近區間為癌化的重要指標。本實驗採集國內瘜肉及大腸癌各時期之樣本，以六個國外常用之APC及p53之突變位置引子，測試其突變，並觀察在蛋白質層面的變化。實驗的目的是希望以DNA定序分析的方法將發生突變的基因篩選出。並在蛋白質的層面，利用二維電泳及質譜分析來探討蛋白質表現之差異。目前，我們由正常及異常組織中發現，有超過30個蛋白質在表現上有增減的差異。其中共有3個蛋白質 ( TSD1、TSD2、TSD3 ) 在癌化演進過程中表現遞減，顯示與癌化程度相關，TSD3蛋白已由質譜分析為與C1q有關之蛋白。然而，由NCBI之Virtual Northern 分析發現此蛋白之mRNA會隨癌化而增高。此外我們發現約有56 % APC及p53突變在癌組織，而此結果顯著低於西方國家之70~85 %，顯示在台灣大腸癌的基因變異情況可能與國外有所差異。

Abstract
The development of colorectal cancer ( CRC ) is believed to follow series progress of pathological changes and with correspondent genetic changes of many genes. This includes intestinal epithelial crypts, aberrant focus, adenoma and carcinoma, each of that commonly involved genetic and proteomic alterations. And in genetic level, it usually includes mutations of APC, p53, K-ras and microsatellite instability. The somatic mutations of APC gene mostly occur in MCR ( Mutation Cluster Region ) in codon 1286-1513. The p53 mutations is dispersed in whole gene with 3 hot spots: codon 175, 248 273. K-ras codon 12 and 13 mutations is preferentially involved in polyps growth of CRC. And microsatellite instability is found in 15-25% CRC patients.

We collect polyps and various stages CRC samples in Taiwan, and design 6 primer pairs of APC and p53 which is widely used in western countries to analyze mutations of the local CRC genetic changes. We also use two-dimensional electrophoresis and mass spectrometry to identify protein expression changes in CRC.

We have found 30 proteins that exhibited either a significant decrease or increase between normal colon tissue and carcinoma, and 3 out of ( TSD1, TSD2, and TSD3 ) these were significantly associated with tumor progression. TSD3 is annotated by mass spectrometry and is identified to be a c1q-related protein. Though there are no report on the function of c1q-related protein, a NCBI virtual northern analysis shows its expression is varied in various cancer. On the other hand, there are only about 56 % genetic changes of APC and p53 during carcinogensis, which is much less than the 70-85 % mutational rate in western CRC patients. It indicates different genetic mutational pattern of CRC in Taiwan.

目 錄
中文摘要…………………………………………………………………………………………I
英文摘要…………………………………………………………………………………………II
前言………………………………………………………………………………………………1
一、大腸癌之簡介 ( Introduction of Colorectal Cancer ) …………………………1
二、大腸癌之細胞分子生物學 ( Molecular Cell Biology of
Colorectal Cancer ) ………………………………………………………………………2
三、大腸癌之基因致癌機轉 ( The Mechanism of Carcinogenesis
in Colorectal Cancer ) ……………………………………………………………………4
甲、APC (Adenomatous polyposis coli)基因…………………………………4
乙、DCC ( Deleted in colon cancer ) 基因…………………………………6
丙、p53 基因………………………………………………………………………6
丁、K-ras (Kirsten-ras gene)基因……………………………………………8
戊、BAT-26 基因 …………………………………………………………………9
四、大腸癌之癌化分期 ( The TNM Staging of Colorectal Cancer ) …………………9
五、大腸癌之臨床症狀及診斷 ( The clinical staging and
diagnosis of Colorectal Cancer )………………………………………………………10
六、大腸癌之專一性抗原 ( The Specific Antigen of Colorectal Cancer ) ………12
甲、血液腫瘤標記常用之癌胚抗原( Carcinoembryonic Antigen；CEA )…12
乙、癌抗原125 ( Cancer Antigen125；CA125 ) ……………………………13
丙、血紅蛋白抗原 ( Hemoglobin Antigen )…………………………………13
七、大腸癌之蛋白質體學 ( Proteomics ) ………………………………………………14
八、大腸癌之腫瘤蛋白質變化 ( Protein Expression in Carcinogenesis
of Colorectal Cancer )……………………………………………………………………16
甲、heat shock 70 蛋白 ( hsp 70 )…………………………………………16
乙、CyclinD1和C-myc …………………………………………………………16
丙、酪氨酸基酉每 ………………………………………………………………16
九、研究目標 (The aims of experimentation )…………………………………………16
材料與方法 ……………………………………………………………………………………19
一、實驗材料來源及製備 ……………………………………………………………………19
甲、實驗藥品 ……………………………………………………………………19
乙、材料來源 ……………………………………………………………………21
丙、材料製備 ……………………………………………………………………21
二、實驗方法 …………………………………………………………………………………21
2-1.基因突變分析…………………………………………………………………………21
甲、從血液中分離白血球 ………………………………………………………21
乙、從白血球中萃取基因體DNA…………………………………………………22
丙、引子( primer )之選擇與設計 ……………………………………………23
丁、聚合酶連鎖反應 ( Polymerase Chain Reaction ；PCR ) …………24
戊、PCR產物的純化………………………………………………………………25
己、自動核酸序列定序 ( Automatic DNA sequencing ) …………………25
2-2. 蛋白質二維電永分析 ( Protein Analysis by Two-Dimension Gel
Electrophoresis ) ………………………………………………………………30
甲、第一維等電點電泳 …………………………………………………………30
乙、第二維SDS-PAGE 展開………………………………………………………32
丙、電泳片染色 ( 銀染 ) ……………………………………………………33
丁、電泳片影像分析 ……………………………………………………………34
戊、In-gel digestion …………………………………………………………34
結果 ……………………………………………………………………………………………37
一、大腸癌患者之基因突變分析 ……………………………………………………………37
甲、APC (Adenomatous polyposis coli)基因 ………………………………37
乙、p53 基因 ……………………………………………………………………38
二、大腸癌患者之蛋白質差異性分析 ………………………………………………………40
甲、資料庫膠片與本實驗膠片之電泳圖比較 …………………………………40
乙、二維電泳圖之區域分析 ……………………………………………………40
丙、大腸癌T1期患者C15之正常及癌化組織中蛋白質差異性比較……………41
丁、大腸癌T3期患者C7之正常及癌化組織中蛋白質差異性比較 ……………42
戊、大腸癌T4期患者C12之正常及癌化組織中蛋白質差異性比較……………42
己、大腸癌不同癌化分期患者之癌化組織中蛋白質差異性比較 ……………42
討論 ……………………………………………………………………………………………45
一、大腸癌突變基因之分子標識 ……………………………………………………………45
甲、APC基因的突變分析…………………………………………………………46
乙、p53 基因的突變分析 ………………………………………………………46
二、蛋白質差異性分析 ………………………………………………………………………47
甲、蛋白質分析之探討 …………………………………………………………47
乙、大腸癌患者之蛋白質差異性探討 …………………………………………49
參考文獻 ………………………………………………………………………………………54

表 ………………………………………………………………………………………………60
表一、大腸癌病患之因之基本資料 …………………………………………………………60
表二、本實驗所用之引子一覽表 ……………………………………………………………61
表三、大腸癌病患之APC-E核酸序列分析……………………………………………………62
表四、大腸癌病患之APC-G核酸序列分析……………………………………………………63
表五、大腸癌病患之APC-H核酸序列分析……………………………………………………64
表六、大腸癌病患之APC-I核酸序列分析……………………………………………………65
表七、大腸癌病患之p53-1核酸序列分析……………………………………………………66
表八、大腸癌病患之p53-2核酸序列分析……………………………………………………67
表九、基因序列分析一覽表 …………………………………………………………………68
表十、經由Mascot及NCBI資料庫比對本實驗之C1q-related factor蛋白質 ……………69
表十一、在不同組織中C1q-related factor蛋白質之表現 ………………………………70
圖 ………………………………………………………………………………………………71
圖一、人體消化系統之腸胃道圖 ……………………………………………………………71
圖二、大腸癌化之腺瘤形成理論 ……………………………………………………………72
圖三、Wnt - signalling pathway …………………………………………………………73
圖四、APC基因DNA序列圖譜 …………………………………………………………………74
圖五、p53基因DNA序列圖譜 …………………………………………………………………80
圖六、蛋白質體學實驗方法 …………………………………………………………………82
圖七、APC-G核酸序列分析 …………………………………………………………………83
圖八、APC-H核酸序列分析 …………………………………………………………………84
圖九、p53-1核酸序列分析 …………………………………………………………………87
圖十、p53-2核酸序列分析 …………………………………………………………………89
圖十一、APC基因之密碼1477位置已有報告指出與大腸癌相關……………………………91
圖十二、SWISS-PROT 蛋白質體資料庫cell line (左)與大腸癌臨床病患C4的
腫瘤組織(右)之二維電泳比較圖 ……………………………………………………………92
圖十三、 C4患者的腫瘤組織二維電泳圖之區域分析………………………………………93
圖十四、T期(C15)正常(左)及腫瘤(右)組織之二維電泳膠片比較圖 ……………………94
圖十五、T3期(C7)正常(左)及腫瘤(右)組織之二維電泳膠片比較圖 ……………………95
圖十六、T4期(C12)正常(左)及腫瘤(右)組織之二維電泳膠片比較圖……………………96
圖十七、不同癌化分期之正常及腫瘤組織之二維電泳膠片I區塊比較圖…………………97
圖十八、不同癌化分期之正常及腫瘤組織之二維電泳膠片II區塊比較圖 ………………98
圖十九、正常(對照，左上)及不同癌化分期之腫瘤組織二維電泳膠片II區塊比較圖 …99
圖二十、不同癌化分期之正常及腫瘤組織二維電泳膠片III區塊比較圖 ………………100
圖二十一、不同癌化分期之正常及腫瘤組織之二維電泳膠片IV區塊比較圖 …………101
圖二十二、正常(對照，左上)及不同癌化分期之腫瘤組織二維電泳膠片IV區
塊比較圖 ……………………………………………………………………………………102
圖二十三、利用輔助雷射脫附游離�飛行時間質譜儀(MALDI-TOF)分析TSD3蛋白質 …103
圖二十四、Mascot Search Results ………………………………………………………104
圖二十五、不同癌化組織檢體之2-DE比較圖………………………………………………105

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連結網址：
1.http://129.85.19.192/prowl/proteininfo.html
2.http://cdnet.stic.gov.tw/
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7.http://tw.expasy.org/sitemap.html
8.http://www.matrixscience.com/cgi/index.pl?page=/search_form_select.html
9.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
10.http://www.sinica.edu.tw/~proteome/
11.http://www.sinica.edu.tw/lib/lsl/ejournal/ej_fc.htm