| 摘要: | 外泌體透過細胞分泌作為功能性蛋白質、mRNA、miRNA、lncRNA、DNA和脂質的載體，並參與細胞間的通訊。近年來，人類血小板裂解液Human Platelet Lysate(hPL)中的外泌體因富含生長因子和細胞因子，已被證實能促進組織修復和再生。此外，其在血液凝固、免疫調節和血管生物學等作用而備受關注。然而，外泌體缺乏標準化的分離和分析方法，進而限制了其在臨床上的應用。本研究的目的在於透過分析血小板裂解液外泌體的內容物，探討與其生物學功能之關聯性，並評估成分之差異，進而了解潛在的調控機制。為了深入探索血小板裂解液外泌體（PLP-EVs）的內容物，本研究採用切向流過濾Tangential Flow Filtration（TFF）進行濃縮和純化PLP-EVs，並嘗試以凍融反應、超音波震盪、磁珠均質化三種不同的方式來打破純化後的PLP-EVs。經過評估我們發現超音波震碎能夠最有效地萃取外泌體內部成分，並進一步分析PLP-EVs內容物成分差異，使用了SDS-PAGE和UPLC-ESI-MS/MS分析系統性地鑒定不同打破方式之外泌體內的蛋白質和脂質及代謝物等物質，為外泌體的藥品化學製造與管制研究建立了基礎。
未來，血小板裂解液（hPL）中的外泌體因其富含生長因子和細胞因子，具有顯著的促進組織修復和再生的潛力，因此在臨床應用中具有廣泛前景。hPL的商品化發展將主要集中在其標準化製備和應用方面。首先，需要建立統一的分離和純化方法，以確保產品的一致性和質量。同時，應進行大量的臨床試驗來驗證其安全性和有效性，特別是在組織工程、再生醫學、免疫調節和血管生物學等領域。隨著技術的不斷進步，hPL產品可望被應用於各種疾病的治療，例如慢性創傷修復、退行性疾病以及免疫相關疾病。此外，hPL外泌體的商品化還需考慮監管合規性和生產成本，以確保其在市場上的競爭力和可及性。總體而言，hPL外泌體具有巨大的臨床應用潛力，未來發展值得期待。
通過本研究，我們不僅加深了對PLP-EVs內容物的了解，還為其臨床應用提供了重要的數據支持和理論基礎，這將有助於推動hPL外泌體在生物醫學領域的進一步發展。
Exosomes are secreted by cells as carriers of lipids, functional proteins, mRNAs, miRNAs, lncRNAs and DNA, playing an essential role in intercellular communication. In recent years, exosomes from platelet lysate have shown their ability to promote tissue repair and regeneration given their abundance of growth factors and cytokines. In addition, they have attracted attention for their role in blood coagulation, immunomodulation, and vascular biology. However, the lack of standardized isolation and analysis methods for exosomes has limited their clinical applications. The aim of this study was to analyze the contents of human platelet lysate exosomes, explore the correlation with their biological functions, and evaluate their differences in composition, with an overarching goal to understand the potential regulatory mechanisms. In order to explore the contents of human platelet lysate exosomes (HPLP-EVs), we used platelet lysate, instead of fetal bovine serum (FBS), and Tangential Flow Filtration (TFF) to concentrate and purify the HPLP-EVs in order to separate the exosomes by size particle. Next, we tried three different ways to break the purified HPLP-EVs: freeze-thaw reaction, ultrasound, magnetic bead beating homogenization. After evaluating the results, we found that ultrasonic is the optimal for extracting the internal components of exosomes, further analyzed the compositional differences of the contents of HPLP-EVs by using SDS-PAGE and UPLC-ESI-MS/MS. We also performed systematic metabolomics characterization of exosomes broken by different methods, and to deepen the correlation between them as a new guideline for future studies, aiming to establish a guideline for future CMC (Chemistry, Manufacturing, and Controls) research on exosomes. |
