膜點狀墨點法於K-ras致癌基因檢測之探討

K-ras致癌基因是目前被發現分佈最廣泛的致癌基因，在多種癌症皆可見其突變情形例如大腸癌、肺癌、乳癌、胰臟癌等。所以國內外醫藥學者無不為此費盡心思，尋找最新、最好的治療方法及藥物來對應，但發現始終缺乏一準確又方便的診斷工具來輔助。有鑑於此在2004年已有學者研發出一全新的K-ras致癌基因診斷平台，盼能解決以上問題，以達早期診斷早期治療之效。此檢測方法可解決傳統診斷侵略性、檢體取得不易等問題，因為過去偵測K-ras突變，多是需先取得病人的癌症組織，再以聚合酶鏈鎖反應(PCR)、直接定序，找出突變點。有此檢測法後，可以最容易取得的血液檢體為檢測目標，直接檢測病人循環系統中K-ras致癌基因的表現情形。當初在研發時是以呈色反應取代螢光反應來判斷結果，其優點在於成本低又具高準確度，但隨著研究的改進，發現呈色法仍容易有人為上的誤差，因此我們這次的研究主要延續此檢測法的開發及改善，我們收集三種癌症檢體參與反應，但將呈色法改為化學冷光法，並對每一基因進行加權計分法。我們得到的結果是這樣的，這種膜點狀墨點法的確可偵測到血液中K-ras致癌基因突變活化的情形，其靈敏度、特異性、分別為95.5%、97.9%，比原來的增加，原來的呈色法其靈敏度、特異性分別為83.7%、90.9%。這種結果啟發了我們繼續研究這個新方法，盼能使其達到更好的境界，成為一準確的診斷工具，能受到世人的信賴，而普遍使用於臨床上，診斷出與K-ras致癌基因有關的癌症，造福更多的人。

K-ras oncogen is frequently found in human cancer. Activating K-ras mutations are present in colorectal adenomas carcinoma、 lung cancer、 breast cancer and pancreatic tumor. Several therapeutic agents have been developed in order to inhibit K-ras activation. However, there is no diagnostic tool available to detect oncogenic K-ras activation in patient，s specimens so far. Therefore, researchers have been developed a new K-ras diagnostic membrane arrays in 2004, trying to resolve the problem. It may monitor and control cancer transformation and malignant progression. In the past, the detection of K-ras mutation was mainly relying on direct sequencing, however the sequencing method could not provide researchers information but mutation sites. Using the new K-ras membrane array can detect circulating cancer cells with mutant K-ras oncogen in peripheral blood. This is a low-cost, convenient, and easy technique for detecting activated K-ras in a small number of circulating tumor cells by the colorimetric membrane array method (CLMA); but the sensitivity still needs improvement. The object of this study is to develop a new platform with chemiluminescence as reporter and weighted values of target genes on the chip in order to achieve a more sensitive, easier to read, and more accurate platform-membrane dot blot on K-ras oncogen detection . In advance, we collected cancer tissues from 220 cancer patients including breast cancer, colorectal cancer, and NSCLC cancer to evaluate the membrane dot blot on K-ras oncogen detection. The results showed the sensitivity of CLMA was fund 83% and the specificity was 96%. On the other hand, the sensitivity of the membrane dot blot on K-ras oncogen detection increase to 95.5% and the specificity increase to 97.9%. In summary, this study has provided a foundation model on which we can develop a variety of diagnostic membrane array for related oncogenes and tumor suppress genes.