旋轉碟盤系統應用於血清中外吐小體之雙水相系統富集法之研究

Abstract

液態生物檢體在近幾年成為熱烈討論的課題，其非侵入式的診斷相較於傳統切片而言為一大優勢。對於未來醫療的發展而言，液態生物檢體更被視為能完成早期檢測、精準醫療與伴隨式診斷的工具。 外吐小體(Exosome)為細胞內吞作用後由細胞膜釋出的奈米囊泡(30-150 nm)，其扮演了細胞與細胞間溝通的媒介。外吐小體內部包含了細胞中的mRNA、miRNA與蛋白質等訊息，接受到這些外吐小體的細胞或環境也許會應而有所改變。近年來越來越多文獻指出外吐小體在臨床應用的重要性，尤其是有關癌症轉移與早期篩檢等研究。也因為如此，外吐小體被認為能擔任液態生物檢體重要的生物標記。 外吐小體之富集一直以來都是一項艱鉅的挑戰，主要原因在於其囊泡的尺度過小。超高速離心法(UC)做為富集方式之黃金準則，但冗長且技術性的操作對使用者而言非常不便。近年來，諸多替代超高速離心的方式與商業化產品如雨後春筍般被開發，低轉速且操作相對快速的方式提供了更多選擇給操作者。但能自動化富集外吐小體之平台卻尚未廣泛使用，通常此類技術需搭配微機電製程，其成本相對高而不易量產。 本研究的主旨在開發一旋轉碟盤平台以提供快速、高產量、純度良好且有自動化潛力之富集法。聚乙二醇(PEG)/右璇糖酐(DEX)之雙水相系統為本論文使用之萃取法。在碟盤設計前，不同高分子聚合物之濃度、靜置時間與執行雙水相分層的次數都會先以Eppendorf操作，並以西方墨點法及奈米粒子追蹤儀進行效果評估與參數選定。測試結果顯示以適當的DEX/PEG濃度組合，無需靜置時間與多次雙水相分層的流程最合適進行碟盤設計，且純度表現也是相對較好的。接著即是著手於碟盤設計與測試，食用色素會配合高分子聚合物進行流體性質的驗證。最後，超高速離心法與三種市售產品─ExoCap、ExoQuick即TEI將與雙水相法(碟盤/Eppendorf操作)進行血清中外吐小體之富集比較。結果的部份將會多加入穿透式電子顯微鏡(TEM)的觀察。實驗結果顯示雙水相法能夠以最快的處理速度(~20分鐘)完成富集，且在純度表現上也是相對優異的。其中以Eppendorf操作雙水相法的純度優於ExoQuickTM (39%)、TEI(55%)。以碟盤平台操作雙水相法則是優於ExoQuickTM(3.3%)、TEI(15%)。另外，在產量的部分雖然ExoQuick表現最好，但雙水相法的產量也還是遠高過超高速離心法與ExoCap。 本研究成功開發一具有自動化潛能之旋轉碟盤系統，該設計能以低轉速且快速的流程完成血清之外吐小體富集。其在產量上優於傳統超高速離心法，且純度表現則是優於市售的商化產品。操作時間與自動化潛能為其最大亮點，相信在未來改善後會更臻完備且能應用於臨床檢測。

Exosomes are nanoscale vesicles (about 30-150 nm) containing proteins and nucleic acids from their parental cell. The recipient cells or the microenvironment will uptake the cargo, hence alterations are made by the exosomes. This effect is believed to have clinical relevance such as cancer metastatic, cardiovascular diseases (CVDs), among others. Therefore, exosomes are promising biomarker for liquid biopsy in the near future. Enrichment of exosome is always a challenging because of their nanoscale feature. Although ultracentrifugation is the gold standard, it is time-consuming and very much operator-dependent. Alternative methods such as commercial kits have been developed, but these are numerous drawbacks. In this thesis, a rapid, high yield and purity exosome enrichment approach is presented. Aqueous two-phase system (ATPS) was utilized to isolate exosomes acquired from serum in about 20 minutes. Three validation methods—western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM)—were used to validate the performance. First, the optimal polymer composition and the protocol were demonstrated by tube-based ATPS method. Using suitable composition with single-stage ATPS and without incubation process is recommended to enrich exosomes acquired from serum. Next, a centrifugal disk was designed for the ATPS enrichment process. The performance was compared with UC, commercial methods—ExoCap, ExoQuick, TEI—and ATPS tube-based method. Data showed that the purity of ATPS method was the best. Comparing with using ExoQuick and TEI method to enrich exosomes, the results of purity by using ATPS tube-based method was higher by 39% and 55% respectively; the results of purity using ATPS disk-based method was slightly higher by about 3.3% and 15%. Although the result of the yield by using ATPS method was not as high as using ExoQuick, the value was preferable to UC and ExoCap method. Operation using ATPS also was the fastest among all methods tested. In conclusion, this thesis successfully enriched exosomes acquired from serum by ATPS, a centrifugal disk platform also was developed to automate the whole process. In the future, a convenient and rapid device may be provided for clinical application.