| 摘要: | 外泌體(exosome)為大小介於30~200奈米間由雙層磷脂質膜所包覆的的囊泡，其內部含有大量的生物標示(biomarkers)如蛋白質、脂質及核酸等，係由細胞吐出於體內循環作為細胞間溝通的橋樑之一，且有許多研究指出外泌體與許多疾病相關，特別是癌症的發生、轉移及擴散，且近年來液態切片(liquid biopsy)於醫學診斷上的角色日趨重要，因此外泌體的分離純化技術顯得格外重要。目前有許多外泌體分離方法超離心、高分子共沈降、免疫親和吸附及超濾法等，然而這些方法往往需要大型儀器或是以離心作為驅動力，使其不易普及，同時離心的步驟也可能導致外泌體在分離過程受到破壞進而導致低回收率，因此本研究致力於提供一以薄膜過濾為基礎的分離程序，由乏血小板血漿 (platelet poor plasma, PPP)中進行外泌體的分離純化，為了有效的提升過濾效能及外泌體回收率，設計一具兩道過濾之分離程序並搭配具有抗沾黏特性的薄膜進行外泌體純化。
第一道薄膜為由聚偏二氟乙烯(PVDF)混摻PS-r-z4vp雙離子高分子的多孔性薄膜，以移除大分子物染物；第二道程序為三醋酸纖維素(CTA)與SBMA水膠之複合膜，用以移除小分子物質並達濃縮效果。經雙離子化的薄膜依其在過濾及抗貼附的效果選出最適化條件Z1及C12-S0.5-N1作為兩道過濾膜進行血漿外泌體的分離純化，經純化分離的外泌體會以奈米粒徑追蹤分析系統(NTA)、酵素結合免疫吸附分析法(ELISA)及場發射掃描式電子顯微鏡(FE-SEM)進行定性定量分析，並與超離心法及商業化之離心超濾法進行外泌體純化之回收率、濃縮倍率及大小分子污染物比例之比較。由實驗結果可得，以雙離子化薄膜所組成的兩道過濾系統可以將外泌體濃縮1.8倍並有效提升回收率達到92%，具有比超離心及離心超濾法更好的回收效率，同時也比較雙離子化前後的薄膜於外泌體分離的差異，由實驗結果可看出以雙離子化改質提升材料抗沾黏表現有效提高外泌體的回收效能。本研究提供了一種新興的純化程序並搭配抗沾黏薄膜的使用有效提升了整體的純化效能，提供未來在外泌體分純化離應用的新選擇。
;Exosomes are small vesicle (30-200 nm) encapsulated by a lipid bilayer which contains biomarkers such protein, DNA, and RNA inside. They play a crucial role in the detection of diseases, cancer progression and metastasis. Due to this, exosome isolation techniques have gained considerable attention, especially when liquid biopsy has played a profound role in diagnosis nowadays. Different isolation methods include ultracentrifugation, polymer precipitation, immunoaffinity capture, ultrafiltration and so on have been proposed and adapted in applications. However, most of these methods require large and expensive instruments. Moreover, centrifugation technique can potentially destroy the exosomes, generating lower recovery. In this regard, we aim to develop an easy and efficient technique by means of membrane filtration to isolate exosomes from HCT116 cell culture medium, and human platelet poor plasma (PPP). Herein, we develop a dual layer membrane filtration process endowed with antifouling property to increase the yield and purity of exosome isolation.
The first layer is a porous membrane made from a blend of polyvinylidene fluoride (PVDF) and zwitterionic polystyrene poly 4-Vinylpyridine (PS-r-zP4VP) that was used to filter out large contaminants. The obtained permeate passed through the second layer based on sulfobetaine methacrylate (SBMA) hydrogel composite cellulose triacetate (CTA) membrane which was utilized to remove smaller contaminants. The final retentate was collected to analyze exosomes. Size distribution and concentration of the retentate were evaluated using NTA, morphology was assessed by FE-SEM, and immunoaffinity of the exosomes were measured by ELISA. Furthermore, protein concentration of the isolate samples was determined through BCA assay to analyze the purity of the exosomes.
The membrane filtration system was able to provide more than 90% exosome recovery with increase exosome concentration by 1.8-fold by comparing with input PPP. In contrast, conventional method using centrifugal ultrafiltration and ultracentrifugation only yielded 60% recovery, 1.2-fold concentrate and 30% recovery, 0.8-fold concentrate. Hence, these results indicate that dual membrane filtration method can provide a promising approach to isolate exosomes in a more economical and efficient manner. |
