血漿中胞外囊泡與親和性聚合物之研究

Abstract

液態活檢為利用偵測血液中的生物標記來反映疾病的關聯性，其中外泌體已成為液態活檢中熱門的檢測標記。外泌體(Exosome)為細胞經由內吞作用後釋出的微小囊泡，粒徑直徑約為30-150 nm，在體內扮演細胞間溝通的橋樑。由於外泌體內含豐富的組成，包括蛋白質、mRNA、miRNA等，可以調控體內微環境，甚至細胞因接收外泌體，改變細胞的狀態，近年來有愈來愈多研究成果支持外泌體與癌症的轉移有高度密切的關係。 許多研究團隊致力於開發能有效地收集血液中外泌體的生物晶片，並藉由靈敏的偵測系統獲取外泌體的訊號。然而，由於血液中含有相當複雜的成分，要從血液樣品獲取足夠代表外泌體的表現量仍是一大挑戰，其中有一很大的原因是血液中的脂蛋白，其粒子的數量遠高於外泌體，因著脂蛋白的數量會降低收集外泌體的效率，使得偵測到外泌體的表現訊號並不顯著，限制了外泌體在臨床測試上的實用性。因此，若能透過對在血液樣品的前處理，去除大部分的脂蛋白，有助於提升偵測到外泌體的表現訊號，對於外泌體應用於臨床檢測上的技術會是很大的突破。 本研究的主旨是利用與脂蛋白有極好親和性的聚合物，用在去除血液中的脂蛋白，進而收集到純度較高的外泌體，達成提升外泌體的偵測訊號。將經過細胞培養並且純化後的外泌體，混合從血液離心後所收集的脂蛋白樣品，並結合與脂蛋白有親和性的聚合物，例如軟骨素、肝素以及葡聚醣，能有效提升外泌體的偵測訊號。同時也與已經有發表去除脂蛋白的試劑與技術進行比較，評估利用親和性聚合物去除脂蛋白的方法，確實具有高效率、操作方便且經濟省時的特點，不僅達成提升外泌體偵測訊號的目的，對於未來在開發相關外泌體的生物晶片，具有非常好的優勢和發展性。

Exosomes are 30-150 nm nano-sized vesicles released from their parental cells into the extracellular space after endocytosis and play a role as mediators in intracellular communication. Exosomes contain proteins, mRNA and miRNA can modulate the microenvironment of the body and even change the behavior of the recipient cells. Recently, many studies have shown a high correlation between exosomes and cancer metastatic. Therefore, exosomes have become an important investigated target of liquid biopsy through blood testing. Many research teams have been devoted to developing effective biochips for capturing exosomes in the blood sample. However, there is still a big challenge to acquire a representative content of exosomes from the blood sample due to the complex components, especially the lipoproteins in the blood. It causes a non-significant signal of the detected exosomes that limits to the use of the exosomal biochip for clinical application. Hence, it is conducive to enhance the exosomal signal of detection through the pretreatment of blood samples such as removing lipoproteins. In this thesis, the goal of using a convenient approach to reduce the lipoproteins in the blood sample for enhancing the exosomal signal by lipoprotein-affinity polymer addition. We prepared the samples of the purified exosomes mixed with lipoproteins which were collected from the plasma sample by ultracentrifugation and combined with lipoprotein-affinity polymers so that can effectively enhance the detected signal of exosomes. Furthermore, to compare with the published tools and the commercial kits using for getting rid of lipoproteins, the polymer-based method is a high efficiency, easy to operate, and time-saving strategy to reduce the content of lipoproteins in the plasma sample and enhance the exosomal signal in the biochip. In conclusion, we successfully enhanced the exosomal signal from the plasma sample in the biochip with the lipoprotein-affinity polymer. A potential strategy by the polymer-based method for removing lipoproteins could be composing advantages and development for the exosomal biochips in the future.