- 學位論文
發展磁珠分離血漿中胞外體內微核糖核酸並應用於心血管疾病之生物標記
Development of Magnetic Bead-based Method for Isolation MicroRNAs in Extracellular Vesicles from Plasma and Assess as Cardiovascular Disease Biomarkers
摘要
近年來胞外體(extracellular vesicles, EVs)由於其獨特的特性如細胞之間的傳遞、信號傳送方式和應用生物標靶於偵測及預後診斷，而在學界引起廣大的興趣。胞外體可攜帶循環微型核糖核酸(microRNA, miRNA)，其為非編碼的短小片段，功能為透過抑制或降解信使核醣核酸(messenger RNA, mRNA)來調控基因表現。而我們的目標是開發一個流程，有別於傳統超高速離心法，改以磁珠分離血漿內胞外體之核醣核酸，並利用心血管疾病的生物標靶（如miR-21，miR-126）來評估心血管疾病的風險。 傳統上，差速超高速離心(differential ultracentrifugation)是廣泛應用於從生物流體收集胞外體的方法，但回收率相對較低，而且產生的極高加速度會導致胞外體降解與破裂且使蛋白質群聚而令分離效果不佳，為了增加胞外體的回收率及效率，我們的解決之道是利用胞外體表面的抗原¬與抗體之間的免疫親和力，將帶有特定抗體(如anti-CD63抗體)的微小磁珠用於辨別及結合目標物，因此，利用磁珠具有高表面積與體積比的特性，能將胞外體及微型核糖核酸有效地從微量血漿樣品中分離出來。實驗結果顯示此流程具有更佳的胞外體以及微型核糖核酸的分離效率，且所需的樣本體積遠小於超高速離心。奈米粒子追蹤儀及穿隧式電子顯微鏡技術用來證明磁珠法能確實抓取胞外體，且純度優於傳統離心法，此外，我們測試EV-miR126-3p與常見心血管疾病之生物標靶cardiac troponin I (cTN-I)和 N-terminal pro-b-type natriuretic peptide (NTproBNP)具有負關聯性，說明此微型核糖核酸具有成為心血管疾病標靶的潛力，最後我們針對不同受試者建立miR-21，miR-126於人類體內的濃度範圍，未來更能應用於開發微流道系統整合與應用，將整套胞外體及微型核糖核酸的抽取系統自動化，並能實際應用於醫療快速檢測上。
並列摘要
Extracellular vesicles (EVs) recently have attracted considerable interests due to their unique characteristics like intercellular communication, signal transporting and potentials as diagnostic and prognostic biomarkers. Circulating microRNAs are short non-coding RNAs that may be actively carried in EVs as signals, which regulate the gene expression through translational repression or mRNA degradation. Our goal is to develop an effective method to isolate EVs and miRNAs from plasma, and further assess their potential application for risk assessment of cardiovascular diseases (CVDs) by utilizing CVD biomarkers, such as miR-21 and miR-126, isolated from EVs. Traditionally, centrifugation is a widely used method to collect EVs from bio-fluids. However, the yield is relatively low. In addition, high acceleration associated with the ultracentrifugation induces the degradation of EVs and the co-precipitation of protein aggregates. In order to increase the yield and purity of enriched EVs, immune-affinity between antibodies and the surface proteins of EVs may be a better solution. We used magnetic beads providing high surface-to-volume ratios with specific antibodies (such as anti-CD63 and anti-CD9 antibodies), which improves the enrichment process. Our experimental results demonstrated the high efficiency in EV enrichment (74%) and miRNA extraction (87%). Supporting results of transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) verified that the bead-based method was able to capture EVs and presented a higher purity than traditional centrifuge-based methods. Further quantifications of EV-miR126-3p and cardiac troponin I (cTn-I) and N-terminal pro-b-type natriuretic peptide (NT-proBNP) in clinical samples revealed negative correlations, suggesting that EV-miR126 may be a potential biomarker for cardiovascular diseases (CVDs). In the future, the developed method could be easily integrated with microfluidic systems such that the entire EV enrichment and miRNA extraction could be automated.
參考文獻
延伸閱讀
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