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研究生:范文君
研究生(外文):Wen Chun Fan
論文名稱:利用survivin抗原與抗體發展免疫分析法以偵測大腸直腸癌
論文名稱(外文):Development of an Immuno-Assay by Using Survivin Antigens and Antibodies for Screening Colon Cancer Patients
指導教授:詹爾昌
指導教授(外文):E. C. Chan
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:67
中文關鍵詞:大腸直腸癌酵素連結免疫吸附分析
外文關鍵詞:ELISAsurvivincolon cancer
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近十幾年來,大腸直腸癌在美國、台灣持續名列十大癌症死因前三名,且發生率與死亡率有持續增加的趨勢,主要原因為大腸癌初期症狀不明顯易造成延遲就醫或誤診;篩檢是降低大腸癌發生率及死亡率最符合經濟效益的方式,因此早期診斷對於大腸癌患者具有相當重要的意義。根據研究指出部分腫瘤相關抗原可以在個體內產生免疫反應,生成具特異性辨認腫瘤相關抗原的抗體。Survivin是屬於IAP家族,具有抑制細胞凋亡功能,先前研究發現survivin在正常人體內幾乎不表現,但在癌細胞中會大量表現,因此發現其對癌症具有特異性。
本研究利用西方墨點法與ELISA偵測大腸癌組織中survivin抗原與抗體表現量。實驗結果顯示西方墨點法可偵測到病患組織中survivin反應(Dukes A:0/1,Dukes B:7/8、Dukes C:6/7與Dukes D:5/6);同時偵測病患血清中anti-survivin抗體,得到敏感度65.8 %(n=205),專一性82.1 %(n=140)。ELISA亦是利用抗原抗體間專一性辨識鍵結的特性來偵測,因此利用survivin重組蛋白質來檢測大腸癌病患血清檢體中的anti-survivin抗體,偵測結果靈敏度為40.3%(n=232),專一性為76.4 %(n=365),若結合anti-survivin抗體與CEA做偵測標記,以任一呈陽性者即為陽性為判別依據下,靈敏度可顯著地提升至64.2 %(p<0.001)。
綜合以上結果,在大腸癌病患組織或血清中,確實可偵測到survivin抗原或抗體;且結合ELISA偵測血清ani-survivin抗體與CEA做為腫瘤標記,偵測靈敏度可顯著地提高,可增加大腸癌篩檢率。
In recent years, incidence and morbidity of colorectal cancer (CRC) is increasing and becoming the third leading cause of cancer death in Taiwan. Because of it’s few and unobvious symptoms, such as blood in or on the stool, pain in the abdomen and bowel habits changes, the patients usually ignore. To date, the diagnostic method for colorectal cancer, including sigmoidoscopy and colonoscopy, are too invasive for most people unwilling to take medical diagnose. Survivin, a member of inhibitor of apoptosis (IAP) family, is a caspase inhibitors. It is broadly expressed in fetal tissues, but is undetectable in the most normal adult tissue. Our purpose is to develop immunoassays by using survivin antigen and antibody for screening cancer patients. We detect the survivin protein and related antibodies response in the tissue and sera from CRC patients by Western Blot and ELISA respectively. Previously, we had detected the survivin expression and found that it is over-expressed in tumor tissues from CRC patients. Furthermore, we detected the survivin autoantibodies response in sera from CRC patients by Western Blot. The sensitivity is 65.9 % (n=205), and specificity is 82.1 % (n=140). In addition, sensitivity of anti-survivin antibodies detected by ELISA is 40.5 % (n=232), and specificity is 76 % (n=365). Combination of anti-survivin antibodies by ELISA and CEA as a multiple-marker significantly promoted the sensitivity to 64.2 %. In conclusion, we develop an efficient immunoassay for screening the CRC patients using multiple-marker of anti-survivin and CEA.
指導教授推薦書
口試委員審定書
授權書………………………………………………………………iii
致謝…………………………………………………………………iv
中文摘要……………………………………………………………v
英文摘要……………………………………………………………vii
目錄…………………………………………………………………viii
第一章 緒論………………………………………………………… 1
1.1 大腸直腸癌簡介……………………………………………… 1
1.2 大腸直腸癌診斷、分期與治療……………………………… 2
1.3 腫瘤相關抗原………………………………………………… 6
第二章 研究目的…………………………………………………… 9
第三章 材料………………………………………………………… 10
第四章 方法………………………………………………………… 13
4.1 重組蛋白質的表現…………………………………………… 13
4.2 兔子多株抗體製備…………………………………………… 17
4.3 兔子多株抗體純化…………………………………………… 18
4.4 組織或細胞內蛋白質的收取………………………………… 19
4.5 西方墨點法……………………………………………………… 19
4.6 ELISA(Enzyme Linkage Immunosorbent Assay)…… 20
第五章 結果…………………………………………………………… 23
5.1 survivin 重組蛋白質純化…………………………………… 23
5.2 西方墨點法偵測大腸癌組織中survivin 表現,並與病患期數
相關……………………………………………………………………… 23
5.3 利用西方墨點法偵測大腸癌血清中survivin 抗體表現……… 23
5.4 利用ELISA 方式篩選大腸癌病患血清anti-survivin 抗體之結
果……………………………………………………………………………24
5.5 其他癌症病患血清中亦可偵測到anti-survivin 抗體…………24
5.6 CEA與anti-survivin 抗體間偵測結果關聯性…………………25
5.7 ELISA 偵測anti-survivin 抗體與CEA 術前值對於臨床病理特
徵分析比較…………………………………………………………………25
5.8 利用ELISA 方式篩選大腸癌病患血清survivin 抗原之結
果…………………………………………………………………………… 27
5.9 利用西方墨點法篩選大腸癌病患血清survivin 抗原之結……… 27
第六章 討論………………………………………………………………… 28
參考文獻……………………………………………………………………… 34
附錄…………………………………………………………………………… 41
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