- 學位論文
乳癌新腫瘤標記之研發
Identification of new tumor markers for diagnosis of human breast cancer
摘要
近年來，乳癌在台灣正逐年增加，是女性第一好發的癌症，同時也高居女性十大癌症死亡原因第四位。研究發現乳癌的早期診斷為降低其發生率及死亡率最有效的方法之一。早期診斷不僅可以大大改善治療的選擇，更可以提高治癒的成功率。乳癌腫瘤標記測定為臨床上最方便簡易的乳癌篩檢方法，但目前所使用之標記都有其限制及缺點。如CEA (carcinoembryonic antigen)、CA15.3 (carbohydrate antigen 15.3)等，其靈敏度及特異性均不佳，所以在臨床診斷上無法達到令人滿意的正確性。因此，本實驗目的為找出具潛力之乳癌腫瘤標記，期望藉由新腫瘤標記配合目前篩檢方法以提高乳癌的診斷率。在吾人的先前研究，利用微矩陣列(microarray )技術，發現有371個基因在三組不同乳癌型態配對組織實驗中，乳癌組織對正常組織均有2倍以上高度表現。本實驗進一步利用生物資訊軟體由371個基因群中挑選出25個基因可能為分泌性蛋白，其中四個基因較具乳癌之特異性，分別為MMP13，CAMP，COL10A1，FLJ25416。收集15對臨床乳癌病人配對組織，進行基因陣列尼龍膜片驗證此目標基因群的過度表現，結果顯示此4個目標基因在癌症組織中確實有過度表現之情形。本實驗首先挑選MMP13進行實驗驗證。收集一系列細胞濃縮上清液，利用MMP13抗體進行西方墨點法實驗，結果發現MMP13確實為分泌性蛋白，具備腫瘤標記之必要條件。進一步以免疫化學組織切片染色證實MMP13蛋白質的確於乳癌組織切片有大量表現情形。根據實驗結果，相信MMP13具有成為新乳癌腫瘤標記之潛力，未來可望應用於臨床以提高乳癌之診斷率。
並列摘要
Within the past decade, the incidence of breast cancer in Taiwan has been rising year on year. Breast cancer is the first most prevalent cancer and the fourth leading cause of cancer-related death among women in our country. Patients diagnosed with early stage breast cancer do not only have a wider range of therapeutic options, but also obtain a higher success rate of therapy than those with advanced stage breast cancer. Tumor markers test is the most convenient method to screen breast cancer. However, detection of tumor markers exhibited certain shortcomings and limitations. For instance, the tumor markers currently available for breast cancer detection include carcinoembryonic antigen (CEA) and carbohydrate antigen 15.3 (CA15.3). Poor sensitivity and specificity greatly limits the diagnostic accuracy of these markers. Therefore, the project mainly aim to identify potential tumor markers for breast cancer. We hope the combined use of new breast tumor markers and routine screening tools can elevate the rate of breast cancer detection. In our previous studies, we have analyzed gene expression profiles in paired specimens of tumor and normal tissue from breast cancer patients of three different cancer cell types by using microarray technology. 371 genes with expression levels in tumor tissue two-fold higher than normal tissue in all three cell type groups were selected. In this study, we pick up 25 genes which maybe encode secretory proteins from 371 genes by employing bioinformatics studies. In the 25 genes, there are four genes MMP13，CAMP，COL10A1 and FLJ25416 which are more specific for breast cancer than other genes. We collect 15 pairs of breast tumor and normal tissue from breast cancer patients, and then find four candidate genes are actually over-expressed in tumor tissues. We selected MMP13 to conduct the following researches first. We prepared cell lines and collected the conditional mediums. Western blot was used to detect MMP13 protein in the culture medium of cell line to confirm that MMP13 is actually a secreted protein. The expression level of MMP13 was also meatured by immunohistochemical stain. We found MMP13 was actually expression in breast cancer tissues. Dependent on the results in the study, we suggest MMP13 has potential to be new tumor marker. We hope MMP13 can be used in clinical diagnosis for elevated the rate of breast cancer detection.