臺灣博碩士論文加值系統

自民國83年開始，大腸直腸癌死亡率已高居第三位，為國人健康的最大威脅。本研究利用蛋白質體學的方式找出相關蛋白去觀察這些蛋白在病人腫瘤與正常組織中的表現情形，以挑選出具有潛力成為生物標誌的蛋白抗原。本研究成功選殖了RPL36，並配合先前的標的RPH3AL、SLP2、p53、survivin、ANXA4和SEC61B進行探討。首先利用Western blot法偵測RPL36蛋白抗體，發現在病人與健檢者血漿中含量有顯著差異(p＜0.001)，敏感度為38.71％而專一性達91.07％。再進一步使用ELISA技術偵測七個不同蛋白在病人及健檢者血液中抗體表現量，經檢測94位病人及75位健檢者，發現靈敏度分別為22.34％、13.83％、36.17％、31.91％、14.89％、11.7％和18.09％，而專一性為96％、90.67％、90.67％、94.67％、93.33％、94.67％和97.33％。以t-test統計結果顯示RPL36、SLP2、p53和SEC61B蛋白在病人與健檢者中抗體表現量有顯著差異(p＜0.05)。再以SPSS software以backward elimination method做進一步分析，發現將SLP2、p53、survivin和ANXA4組合後發現其靈敏度為52.1％，專一性為88％，且AUC提升為0.754，故結合後的分析效果較佳。綜合以上結果，期望能藉此研究發展出一個多重抗體組合的檢驗試劑，藉由快速而經濟的檢測方式，以有效達到早期篩檢、早期治療的目標。

Colorectal cancer (CRC) has become the third leading cause of death since 1994. This study used proteomics technologies to find candidate proteins and observed their expression in both the tumor and normal tissues. Based on that observation, we discovered several proteins that are potential biomarkers. This study successful cloned the RPL36 and discussed it along with the previous targets including RPH3AL, SLP2, p53, survivin, ANXA4 and SEC61B. Firstly, we used western blot to detect RPL36 autoantibody and found a significant difference between CRC patients and volunteers regarding RPL36 autoantibody in the plasma (p<0.001), with a 38.71% sensitivity and a 91.07% specificity. Secondly, we used ELISA to detect those proteins’ autoantibody response in the blood of patients and volunteers. In the ensuing screening of 94 CRC patients and 75 volunteers, we found that the sensitivity of RPL36, RPH3AL, SLP2, p53, survivin, ANXA4 and SEC61B were 22.34%, 13.83%, 36.17%, 31.91%, 14.89%, 11.7%, 18.09% and the specificity were 96%, 90.67%, 90.67%, 94.67%, 93.33%, 94.67%, 97.33%, respectively. The t-test statistics showed a significant difference between patients and volunteers (p<0.05) regarding RPL36, SLP2, p53 and SEC61B autoantibody response. A detection using SLP2 combined with p53, survivin and ANXA4 autoantibodies produced an increase in sensitivity (52.1%), specificity (88%) as well as AUC (0.754), indicating the improved results of such a combination. In summary, this study will hopefully develop a screening test based on a combination of multiple biomarkers response for the monitoring or diagnosis of CRC.

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
目錄 vii
附圖目錄 xiii
附表目錄 xvi
第一章 緒 論 1
1.1大腸直腸癌簡介 1
1.1.1大腸直腸之解剖生理 1
1.1.2大腸直腸癌症狀 2
1.1.3大腸直腸癌病因 2
1.2大腸直腸癌的診斷與治療 2
1.2.1大腸直腸癌分期 2
1.2.2大腸直腸癌診斷方式 3
1.2.3大腸直腸癌的治療 4
1.3目前常用之腫瘤標記 5
1.3.1 Fecal markers 5
1.3.2 Serum or blood markers 5
1.4候選基因 6
1.4.1 Claudin 3 (CLDN3) 6
1.4.2 Ribosomal Protein L36 (RPL36) 6
1.4.3Rabphillin 3A-like (RPH3AL) 7
1.4.4 Stomatin-like 2(STOML2、SLP2) 7
1.4.5 Baculoviral IAP repeat containing 5（BIRC5、survivin） 8
1.4.6 Tumor protein p53（TP53、p53） 8
1.4.7 Annexin A4（ANXA4） 9
1.4.8 Sec61 beta subunit（SEC61B） 9
第二章 研究目的 10
第三章 實驗材料 11
3.1洋菜膠電泳相關溶液 11
3.2細菌培養相關溶液 11
3.3 西方墨點法相關溶液 12
3.4蛋白質純化相關溶液 14
3.5 ELISA相關溶液 15
3.6 多株抗體製備相關溶液 16
第四章 實驗方法 18
4.1人類大腸直腸癌CLDN3、RPL36 gene之選殖 18
4.1.1大腸直腸癌細胞株（CoLo205）之細胞培養 18
4.1.2由CoLo205細胞株中萃取Total RNA並測定濃度 18
4.1.3反轉錄（Reverse transcription reaction）與聚合酶連鎖反應（Polymerase chain reaction） 19
4.1.4洋菜膠電泳（Agarose gel electrophoresis） 20
4.1.5洋菜膠電泳回收PCR產物（Gel/PCR DNA fragments extraction） 20
4.1.6 PCR產物與基因選殖載體的接合反應（Ligation）20
4.1.7 LB培養液的製備 20
4.1.8勝任細胞之製備（Competent cell preparation） 21
4.1.9質體轉型（Plasmid transformation） 21
4.1.10轉殖基因確認 22
4.1.11將目標基因送至表現載體中並確認 22
4.2重組蛋白（recombinant protein）之表現與純化 22
4.2.1誘導重組蛋白表現 22
4.2.2純化重組蛋白 23
4.2.3透析（Dialysis） 24
4.2.4蛋白質定量分析 24
4.3大腸直腸癌專一性抗體之測定 25
4.3.1西方墨點法（Western blot） 25
4.3.2酵素連結免疫吸附分析（ELISA） 25
4.3.3統計分析 26
4.4 抗體titer之測試 26
4.5 多株抗體之製備 27
第五章 結果 29
5.1 Candidate gene的選定 29
5.2篩檢大腸直腸癌病人及健檢者血漿之CLDN3抗原反應 29
5.3基因選殖與蛋白表現 29
5.3.1 CLDN3、RPL36之DNA片段表現 29
5.3.2 PCR產物與基因選殖載體的接合反應（T-A clone）與確認 30
5.3.3轉殖基因至表現性載體及菌體中 30
5.3.4重組蛋白之表現、純化與透析 31
5.4重組蛋白之確認 31
5.5用western blot偵測RPL36在血漿中之抗體反應 31
5.6利用ELISA偵測不同蛋白在血液中之抗體反應 32
5.6.1對RPH3AL抗原的反應 32
5.6.2對RPL36抗原的反應 33
5.6.3對SLP2抗原的反應 33
5.6.4對p53抗原的反應 34
5.6.5對survivin抗原的反應 34
5.6.6對ANXA4抗原的反應 35
5.6.7對SEC61B抗原的反應 35
5.6.8大腸直腸癌、其他癌症病人與多重抗原組合之評估 35
5.7 RPL36多株抗體之製備 36
第六章 討論 38
參考文獻 43
附錄 49
附圖 49
附表 72
Supplementary data 80


附圖目錄
圖一、以western blot鑑定病人血漿中之CLDN3表現 49
圖二、primer設計 50
圖三、大腸直腸癌細胞株經PCR放大之基因片段 51
圖四、用限制酶切割鑑定選殖基因(CLDN3、RPL36) 52
圖五、用限制酶切割帶有基因的pGEM-T easy載體與pET29b載體 53
圖六、轉殖基因至表現菌體中之確認 54
圖七、利用E. coli BL21（DE3）表現CLDN3與RPL36蛋白 55
圖八、純化後之重組蛋白 56
圖九、重組蛋白之確認 57
圖十、以western blot鑑定大腸直腸癌病人與健檢者血漿中RPL36 autoantibody表現 58
圖十一、以western blot方式大量篩檢大腸直腸癌病人與健檢者血漿中RPL36 autoantibody表現 59
圖十二、以ELISA偵測血漿中自體抗體反應之format 60
圖十三、不同重組蛋白經純化與透析後之跑膠圖 61
圖十四、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對RPH3AL autoantibody反應 62
圖十五、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對RPL36 autoantibody反應 63
圖十六、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對SLP2 autoantibody反應 64
圖十七、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對p53 autoantibody反應 65
圖十八、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對survivin autoantibody反應 66
圖十九、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對ANXA4 autoantibody反應 67
圖二十、以ELISA方式大量篩檢大腸直腸癌病人、健檢者與其他癌症病人血液中對SEC61B autoantibody反應 68
圖二十一、以receiver operating characteristic（ROC） curve觀察不同重組蛋白在血液中之抗體反應 69
圖二十二、RPL36抗體的確認 70
圖二十三、抗體純化後的確認 71


附表目錄
表一、Candidate gene library 72
表二、實驗所用之primers 73
表三、偵測大腸直腸癌病人對七種抗原反應之靈敏度與專一性評估 74
表四、分期探討大腸直腸癌病人與健檢者血液中autoantibody表現 75
表五、詳細比較大腸癌病人對於不同抗原反應之分析結果 76
表六、其他癌症病人檢體對七種大腸癌抗原之反應 79

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