以酵素免疫分析法探討腫瘤相關抗原之自體抗體作為肺癌及食道癌腫瘤標記之可行性

肺癌占台灣地區總人口所有癌症死因之首位，而在男性所有癌症死亡原因方面，肺癌及食道癌則分別佔第二位及第六位，研究台灣地區肺癌及食道癌發生的原因，並尋找早期偵測及預後的指標，實為一項刻不容緩的工作。腫瘤抗原可分為腫瘤特異性抗原（tumor specific antigen, TSA）和腫瘤相關抗原(tumor associated antigen, TAA)，其中腫瘤相關抗原TAA在腫瘤細胞的表達量遠超過正常細胞。有文獻指出利用SEREX（serological identification antigens by recombinant expression cloning）可以檢測出在癌症患者的血清中有抗體可以辨識腫瘤相關抗原(tumor-associated antigen)，像是調控細胞增生的蛋白，如Ras、CSK、Erbb2；mRNA 結合蛋白，如p62、IMP1；和一些調控細胞週期的蛋白，如Cyclin B1、p16等，大都因為癌症而導致突變或不正常的表現，因此偵測這些自體抗體的腫瘤抗原可成為在腫瘤形成時偵測不正常的細胞機制的標的。本論文製備純化出7個腫瘤相關抗原候選蛋白：IMP1、Cyclin B1、Ras、CSK、Erbb2、p90和p16，針對這些蛋白的部分胺基酸序列所做的Trx重組融合蛋白，在其NH2-端接上六個His-tag，故以鎳離子親和性管柱純化，再以膠層過濾層析法、硫銨沉澱進行純化及濃縮。以純化的TAA當抗原，再以ELISA檢測30位正常人、60位肺癌及60位食道癌患者血清中的TAA抗體。結果發現在肺癌部份，統計上以p90、p16在癌細胞型態、分化程度等分類都有顯著差異，其中又以p16的整體出現率高達21.7％。在食道癌部份，IMP1的整體出現率約20％，而以癌細胞分類時鱗狀細胞癌出現率較高，用癌症期數分類時，則癌化Ⅱ、Ⅲ期出現率較其他高，還有以分化程度分類時中等偏不佳有較高的出現率。這些結果表示p90、p16、IMP1可能適合作為肺癌及食道癌的免疫診斷標誌。

The lung cancer places the lead cause of death of cancer in Taiwan area, and in male of all cancer, lung cancer and esophagus cancer is the second and the sixth, respectively. To study the mechanism of tumorigenesis of lung and esophagus in Taiwan, and to search the marker which can be detected in early stage and prognostic indicator is very important. The antigen of tumor is divided into tumor specific antigen (TSA) and tumor associated antigen (TAA), the expression of TAA in tumor cells is higher than in normal cells. Previous study used the SEREX (serological identification antigens by recombinant expression cloning) and find that antibody in the serum of cancer patients can recognize TAA. For example, the proteins of regulating cell proliferation, such as HER / neu, Ras, CSK, Erbb2; the mRNA binding proteins, such as the p62, IMP1; and the proteins of regulating cell cycle, such as cyclin B1, p16, most of them whose mutation or aberrant expression could result in cancer formation. Therefore, the TAAs against these autoantibodies can be the markers to detect the cellular abnormal mechanism when tumoriogenesis. In this study, we constructed and purified 7 candidate TAA : IMP1、Cyclin B1、Ras、CSK、ERbb2、p16 and p90. The recombinant proteins, which were designed according to their amino acid sequences, were linked to 6 His-tag in their N-terminal, then we used Ni-NTA affinity chromatography, gel filtration chromatography, and ammonium sulfate precipitation to purify and concentrate candidate TAA proteins. The purified recombinant TAA as the antigen, we examine the expression of TAA in the sera from 30 normal males, 60 male lung cancer patients and 60 male esophagus cancer patients by ELISA. In lung cancer, we found there are significant differences in p90 between normals and patients with different cancer cell types, stage and differentiation levels, etc. We also observed the same results in p16 which whole frequency of positive is 21.7%. In the esophagus cancer, the whole frequency of positive of IMP1 is about 20%, between normals and squamous cell carcinoma compared with other cell types, cancer stage II and III compared with other stag, and in the differentiation levels of middle - poor level, IMP1 has higher frequency. These result mean the p90, p16, the IMP1 may suit to be used as the immune marker to diagnose the lung and esophagus cancer.