外泌體前處理以減少樣品雜質之研究

Abstract

隨著癌症檢測技術的進展，新興的液態生物檢體技術有別於過往的活體組織切片技術，液態生物檢測方式是屬於非侵入式，透過檢測生物體液中的生物標記來反映疾病的狀況，目前許多研究指出血液的偵測可以在癌症與相關疾病發揮作用，因為血液中富含細胞吐出的外泌體。然而，目前面臨的最大挑戰是血液中的脂蛋白，檢測時脂蛋白會阻擋外泌體訊號，使得偵測到外泌體的訊號並不顯著，限制了外泌體在臨床測試上的實用性。 本研究的目標是研發具備臨床研究潛力的去除樣品中之雜質以達到外泌體純化方法。使用的方法為，利用親水與疏水端對樣品中之雜質進行分解。結果顯示前處理和密度梯度超高速離心法(DGUC)兩者實驗的搭配，可去除部分的樣品中之雜質，進而萃取純度較高的外泌體。並且，發現將經過細胞培養且純化後的外泌體與添加物反應，進而可以萃取出外泌體特點蛋白。初步結果指出本研究有助於辨識病人的外泌體訊號，但是仍然需要更多的測試才能確實佐證未來的檢測潛力。

The emergence of liquid biopsy offers a promise to advance non-invasive diagnosis in comparison to traditional tissue biopsy. Therefore, many clinical trials have shown that blood samples can play a role in most solid tumors due to the abundance of components generated by tumor cells and arrived at the circulation, such as exosomes. However, lipoprotein can cause significant issue in hampering effective isolation of exosomes rendering realization of exosomal signal towards liquid biopsy challenging. The main goal of this thesis was to reduce sample impurities in order to improve exosomal signal. Towards this end, properties of hydrophilic head and the hydrophobic tail were utilized to decompose impurities in samples. Results indicated that the combination of reagent and density gradient ultra-centrifugation (DGUC) experiments removed most impurities, thereby extracting exosomes with high purity. Moreover, it was found that the approach enabled intracellular proteins from purified exosomes from cell-cultured medium. Therefore, the current approach seems to be able to treat impurities, which were helpful to detect the exosomal signals of patients. Additional data is needed to realize the full potential of this approach for future use.