本實驗利用Molecular beacon與細胞中microRNA hsa-miR-10b進行雜合反應，及利用HILO螢光顯微鏡系統提高靈敏度，藉由影像圖直接區分hsa-miR-10b於不同肝癌細胞中的表現量，結果與反轉錄即時定量聚合酶連鎖反應（RT-qPCR）之實驗結果相符，甚至克服RT-qPCR於低濃度下（< 100 fM）可能造成誤判的問題。最後透過兩段不同的Molecular beacon同時標記hsa-miR-10b與U6 snRNA，並以多光源同時偵測，成功以螢光原位雜合法結合HILO螢光顯微鏡相對定量肝癌細胞中microRNA hsa-miR-10b。此方法不須透過任何萃取步驟及序列複製放大，可藉由影像分析microRNA的表現量，能將其應用於癌症檢測及分期上。論文第二部分為發展以毛細管電泳暨雷射誘發螢光分離染色體之方法。利用秋水仙素（Colcemid）使大部分的細胞停留於分裂中期（Metaphase），於顯微鏡下觀察並以虹吸進樣的方式將單細胞引入毛細管內，利用Lysis Buffer消化細胞膜並分離染色體。透過酸性甲醇固定細胞，並以超音波震盪於影像圖中可清楚觀察到單條染色體，也成功利用螢光探針標記一號染色體。期望未來能夠將染色體與核碎片分離，使我們透過電泳準確的分離特定染色體。 In this study, using the molecular beacon for microRNA hsa-miR-10b hybridization in cells, and improving the sensitivity by Highly inclined and laminated optical sheet (HILO) microscopy. We could distinguish the expression of hsa-miR-10b from different images of hepatocellular carcinoma cells. These results are in agreement with the reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR), overcoming the problems from miscarriage of low concentrations (< 100 fM). Finally, we simultaneously detect hsa-miR-10b and U6 snRNA by two different molecular beacon and multiple sources. We are able to relative quantification of hsa-miR-10b from hepatocellular carcinoma cells by fluorescence in situ hybridization combined with HILO microscopy. The method does not need any extraction step and sequence replication. It could be applied to detect and stage of cancer by analyzing expression of microRNA from different images. The second part is focus on the separation of chromosomes by capillary electrophoresis with laser-induced fluorescence. We are able to make the cells remain in metaphase by treating colcemid. Observing on the microscope and injecting a single-cell into the capillary by siphon injector way. Then, using Lysis Buffer digest the cell membrane and separate chromosomes. We could clearly observe a single chromosome on image by ultrasonic vibration the cells of fixing in methanol with acetic acid. We are able to labeled chromosome 1 by fluorescent probe. We hope that we could separate chromosomes and nuclear debris in the future, so that we can accurately analyze specific chromosome by electrophoresis.
