外泌體包裹載體的製備及凍乾保存

Abstract

隨著液態生物檢體於臨床診斷技術持續地發展與進步，近幾年在研究血液中的循環腫瘤細胞(CTC)、循環腫瘤DNA(ctDNA)、外泌體(Exosomes)領域受到格外重視。外泌體是藉由細胞內吞作用所釋放具有脂質雙層膜結構的奈米囊泡，其中包含親代細胞的mtDNA、miRNA、蛋白質和訊息因子等物質，它們可以做為信號傳送給其他細胞或環境以改變其功能，也能做為生物標記物來進行檢測與分析，更由於其優異的生物相容性與膜表蛋白的靶向性，使得外泌體具有高度的潛力作為包覆載體的發展。然而，外泌體用於臨床的使用上，仍然面對許多的困境，除了保存溫度的限制，導致外泌體的保存與運輸不便之外，在臨床的檢測方面，期待在捕獲血液中的外泌體的同時，進行特定蛋白的定量分析。 本論文主旨為以外泌體作為載體來開發出蛋白質定量技術以及改善外泌體保存溫度的限制。透過擠壓的方式成功且高效率地使特定的重組蛋白包覆其中，使用已開發出的外泌體檢測晶片，判斷重組蛋白成功包覆在外泌體的依據。此外，使用冷凍乾燥的技術對外泌體進行保存，對外泌體進行冷凍乾燥的處理，並將凍乾的外泌體保存在不一樣的溫度中，並且在不同的時間點與保存在-80°C的外泌體進行膜內蛋白活性的比較，探討外泌體經過凍乾後，抗凍劑對於其膜內蛋白質活性保護的效果，以及不同保存溫度與保存週期對於外泌體的影響。 本研究成功地展示透過擠壓方式將外泌體作為載體，包覆特定的重組蛋白，並且透過外泌體檢測晶片中進行偵測與分析，同時找出外泌體在冷凍乾燥後保存條件，有效地改善了外泌體保存的限制。未來可應用在外泌體的特定蛋白定量分析，並且能透過冷凍乾燥的技術進行樣品的運送及保存。

Applications of liquid biopsy in clinical diagnosis with the continued development and advancement in recent years, many clinical trials performed using human blood samples, including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes. Exosomes are produced by cells through the endocytosis process. They are lipid bilayer particles composed of heterogeneous components, such as DNA, miRNA, and proteins. These derived vesicles play important roles in intracellular communication of various types of cells and regulating the physiological process. Exosome can be a good candidate for a biological carrier due to its excellent biocompatibility and targeting ability. However, there are lots of challenges about exosomes storage owing to the preservation temperature that restricted exosome samples handling and transportation. In clinical detection, it is expected to perform exosome-based protein quantification and analysis while capturing exosomes in the blood. In this thesis, we used exosome as a carrier to load recombinant proteins for exosome-based protein quantification and evaluated the exosomes preservation. We used extrusion method to load the targeting recombinant proteins into exosomes and identified the results by exosome-affinity chip. Additionally, we used lyophilization method as a preservation strategy for exosomes storage. Using cryoprotectants to investigate the activity of lyophilized exosomal proteins at different temperatures and periods comparing with the exosomes which were stored at -80°C. Herein, we successfully demonstrated the loading cargo into exosomes with recombinant proteins by extrusion. We detected and analyzed these carriers using exosome-affinity chips. Furthermore, we optimized the preservation conditions for lyophilized exosomes. In the future, exosomal carriers will expect to perform the exosome-based protein quantification and to implement the preservation and transportation of the samples efficiently by lyophilization.