人類大腸直腸癌之病發率高居台灣十大癌症死因第一名，且病例有逐年上升趨勢。目前醫療上對於大腸直腸癌缺乏有效的早期檢測方法，使之大腸直腸癌病人無法被早期診治。目前研究指出，一些腫瘤相關抗原會對不同癌症病人自身自體免疫抗體產生反應。故本研究目的希望偵測大腸直腸癌病人血清中自體抗體與組織之抗原表現，以進一步評估其靈敏度、專一性與表現率並用於癌症診斷上。本研究初期挑選出11個大腸直腸癌之候選相關抗原用來偵測病人血清中的抗體反應，並依據初步偵測結果將研究主軸集中於Annexins此抗原。初步推測大量表現Annexins可能會誘發自體抗體反應，而尚未有研究證實可從癌症病人血清中測得抗體反應。研究中以Annexins重組蛋白作為抗原並利用酵素連結免疫試驗(ELISA)與西方墨點法(Western blotting)分別偵測220個不同Dukes’期數之大腸直腸癌病人及216個健康捐贈者血清中對於Annexins的自體抗體反應，同時依據ELISA結果訂定出適合之cut-off值。研究結果顯示，大腸直腸癌病人與健檢者血清中具顯著差異性的自體抗體反應，其靈敏度與專一性分別為68 % 與 74 %。其靈敏度更可藉由結合臨床CEA數值(CEA > 5 ng/ml) 達到83 %，其中抗體反應比率為Dukes’後期高於Dukes’前期。另外利用Annexin單株抗體以螢光免疫染色法(IHC)與西方墨點法(Western blotting)方法偵測60對大腸直腸癌病人組織中Annexin表現情形。結果顯示有45 % 病人組織中其抗原上升表現率高於兩倍，同時癌組織亦較正常組織具有高度表現性(P < 0.001)。故藉由研究偵測Annexin自體抗體反應與表現情形，可作為大腸直腸癌在臨床診斷與癒後評估上的另一個參考指標。

Human colorectal cancer (CRC) has been listed as one of the most present cancer in Taiwan. According to recent studies, some cancer patients possessed autologous antibody could recognize different tumor-associated antigens (TAAs). Therefore, the aim of this study was to detect auto-antibody and antigen in the CRC patient’s serum and tissue, then measure their sensitivity, specificity and prevalence for cancer diagnosis. In the initial investigation, we seleted eleven TAAs to detect their corresponding antibodies in patient’s serum and finally we focus on Annexins. Here, we are the first to demonstrate that over-expression of Annexins presumably induces auto-antibody response. We have examined 220 sera samples from CRC patients and 216 healthy volunteers’ samples to observe and correlate the autoantibodies response to Annexins in different Dukes’ stages of CRC. By using Western blot and ELISA analysis with recombinant Annexins as antigen and an appropriate cut-off point, we demonstrated that a high level of Annexins autoantibodies was more significantly presented in CRC patients than healthy controls with a 68 % and 74 % of sensitivity and specificity, respectively. In addition, the diagnostic sensitivity could be reached to 83 % determined by either positive antibody response or level of CEA (CEA＞5 ng/ml). We also found Annexins autoantibody response occurred more frequently in Dukes’ advanced stage than early stages of CRC. Alternatively, we also detected the expression of Annexin in 60 tissue pairs of CRC patients by IHC and Western blotting. Our finding revealed that the Annexin protein level was relatively up-regulated (expression ratio > 2) in 45 % CRC patients. Such result demonstrated significantly higher level of Annexin protein expression in tumor tissues than in normal tissues (P < 0.001). In conclusion, the measurement of anti-Annexin autoantibodies response and Annexin expression could be an alternative approach and useful diagnostic and prognostic method for CRC patients.

指導教授推薦書
口試委員會審定書
長庚大學碩士論文著作授權書…………………………………..………............iii
誌謝…………………………………………………………………......................iv
中文摘要…………………………………………………………….......................v
英文摘要……………………………………………………………......................vi
縮寫表…………………………………………………………………………….vii
目錄………………………………………………………………………………..ix
附圖目錄………………………………………………………………………….xii
附表目錄…………………………………………………………………………xiv
第一章 研究動機與背景介紹 ……………………………………………….......1
1.1 研究動機……………………………………………………………..1
1.2 人類大腸直腸癌(Human Colorectal Cancer)……………..………...3
1.2.1 大腸直腸癌的盛行狀況……………………………………….3
1.2.2 大腸直腸癌病因……………………………………………….3
1.2.3 大腸直腸癌的病狀…………………………………………….5
1.2.4 大腸直腸癌的診斷…………………………………………….6
1.2.5 大腸直腸癌的分期…………………………………………….8
1.2.6 大腸直腸癌的治療…………………………………………...12

1.3 大腸直腸癌之癒後(Prognosis)…………………………..................15
1.4大腸直腸癌之腫瘤抗原(Tumor-associated antigens-TAAs) ……....17
1.5大腸直腸癌對抑癌藥物之低治療率……....……………..................19
第二章 材料與方法……………………………………………………………...21
2.1 實驗材料………………………………………................................21
2.2 實驗方法………………………………………................................23
2.2.1 大腸直腸癌抗原基因之選殖………………………………...23
2.2.2 重組蛋白(recombinant protein) 之表現與純化……………..31
2.2.3 酵素連結免疫試驗(ELISA)………………………………….34
第三章 實驗結果………………………………………………………………...36
3.1 Proteomics方法尋找大腸直腸癌之腫瘤相關抗原………………...36
3.1.1 Proteomics方法找尋大腸直腸癌組織up-regulated protein…36
3.1.2 腫瘤相關抗原之基因轉殖…………………………………...37
3.2 表現選殖基因之重組蛋白與純化……………................................39
3.2.1 基因選殖重組蛋白之表現…………………………………...39
3.2.2 純化基因選殖之重組蛋白…………………………………...40
3.3 篩檢大腸直腸癌病人及健檢者血清之抗原抗體反應……………42
3.3.1 建立血清抗原抗體反應之Western blot format……………..42



3.3.2 大量以Western blot方法偵測大腸直腸癌病人與健康者 血清對Annexins之自體抗體反應……………………………...43
3.3.3 建立偵測血清抗體反應之ELISA format同時大量篩檢大腸直腸癌與健康者血清對Annexins之抗體反應…………....45
3.4 評估其他癌症病人血清對Annexins之抗體反應……………........47
3.5 偵測大腸直腸癌病人組織中Annexin之表現情形……………….48
3.6 偵測大腸直腸癌病人血清中Annexin之表現…………………….50
第四章 研究討論………………………………………………………………...51
第五章 結論….…………...…………………………….………………………..53
參考文獻…………..……………………………………………………………...55
附錄……………………………………………………………………………….72
附圖…………………………………………………………………………….72
附表…………………………………………………………………………….91
Supplementary data……..……………………………………………………….104

行政院衛生署 - http://www.doh.gov.tw/statistic 。
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