臺灣大學: 應用力學研究所胡文聰楊正偉Yang, Cheng-WeiCheng-WeiYang2013-03-212018-06-292013-03-212018-06-292010http://ntur.lib.ntu.edu.tw//handle/246246/250034癌症是現代人主要的死亡原因之一，其中約９０％癌症病患的死亡原因是來自於轉移性的癌症 [6]，轉移性癌症是腫瘤釋放循環腫瘤細胞 (circulating tumor cells(CTC)) 到病人的血液或淋巴管中，經由血液或淋巴循環系統散布到身體其他器官，並在健康器官中形成新的腫瘤，而目前的醫學影像和血液中的癌症相關抗體診斷在初期的轉移癌症中是難以檢測。所以在臨床診斷中，能提供醫生病人血液中的CTC轉移的即時情況極為重要，能作為治療效果的評估和治療過程的監視。然而CTC在血液中的數量極為稀少，每十億血球細胞中只有數個CTC，因此快速且廉價地分離和偵測血液中的CTC為工程和醫學上的重要的挑戰。 本研究提供一具有微流體結構的離心碟盤系統設計，利用免疫磁珠分離法抓取CTC，並用多個免疫螢光標定來判斷抓取到的細胞。目前我們以人類乳腺癌細胞株 (MCF7) 模擬病人血液中的稀少CTC，並用健康人的全血來模擬病人血液的背景細胞及與台大醫院合作實際偵測乳癌病人之CTC數(詳細資料附於Appendix 2)。MCF7與Anti-EpCam-PE和 Anti-PE BD magnetic beads進行結合，使其能被碟盤上的磁鐵陣列抓取，抓取後在碟盤上進行Anti-cytokeratin-FITC 和 Hoechst33342兩個螢光抗體標定，作為螢光顯微鏡下判斷CTC的螢光信號。為了能在碟盤上進行多個螢光標定，設計出氣孔閥 (Vent valve) 作為多個螢光染劑和清洗溶液的序列式開關，並利用氣孔控制盤 (Vents control plate, VCP) 來同步控制各個對應的氣孔閥門，且此閥的操作在未來是有可能完全自動化並整合在微流碟盤中，減少人為操作的實驗誤差。 實驗結果顯示，在１毫升的健康人全血中混入50~300個 MCF7的實驗中，均有50~60%的回收率，而一片碟盤一次的處理速率為每小時２毫升血液甚至可以再增量，且此技術的靈敏度可達大於10-7。因此系統有高的血液處理速率、操作簡單、低成本的材料和加工、細胞損失率在可接受範圍並且有機會自動化減少人為操作成本和誤差。可望未來能經由病人的臨床結果建立起此系統的標準化並提供醫生良好的診斷工具。Capture and detection of specific rare cells have shown great promise for biological and clinical studies. Circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients represent a potential alternative to invasive biopsies as a source of tumor tissue for detection, characterization, and monitoring of non-haematologic cancer. This thesis outlines a novel microfluidic device to capture and detect CTCs in patient blood. As proof-of-concept, experiments were conducted where a cell line of MCF7 was used to simulate CTCs and healthy whole blood was used for background peripheral blood. A continual flow process via a centrifugal microfluidic disk platform to capture MCF7 in blood immunomagnetically and enumerate them on-disk with a complete batch process of multi-fluorescence labeling is presented. The MCF7 are labeled with anti-EpCAM-PE and anti-PE magnetic beads for magnetic force capturing and with anti-cytokeratin-FITC antibodies and Hoechst33342 for detection. In order to allow precise timing in liquid delivery during the multi-fluorescence labeling processes, on-disk deterministic vent valves were designed. To characterize the disk performance of target cell capturing and fluorescence labeling, three different labeling procedures were used. Results show that the cell-capture yield of the disk was about 65% and the throughput was 2ml/hr or more. After staining two-label fluorescence on the disk, the yield was around 50%. The sensitivity of the technique in enriching rare cells from whole blood (>1ml) is up to 10-7. Direct fluorescence labeling on the disk without sample transfer and manual operation greatly helped to reduce cell loss. The total procedure, from magnetic bead labeling to completing two-label fluorescence staining, takes place within 1.5 hours. In order to determine the efficiency of the disk in enumerating CTC from patient with epithelial cancers, the breast cancer data of 14 women were collected (as shown in Appendix 2). Advantages of the present platform include simple operation, high throughput, an acceptable level of cell loss, and a potentially low system cost, which should substantially ease the effect in cyto-analysis of rare cells.2369457 bytesapplication/pdfen-US稀少細胞微流系統癌細胞碟盤離心MCF7rear cellmicrofluidicspositive selectionCTCdisk[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/250034/1/ntu-99-R97543042-1.pdf