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外泌体(exosome)是细胞产生的胞外囊泡的一种,在20世纪80年代末首次被描述[1-2]。外泌体作为一种细胞间近距离通讯的特殊介质,在包括免疫反应、抗原递呈和信号转导[3]等各种生理过程中发挥着重要作用。几乎所有真核细胞都可以分泌外泌体,包括脂肪细胞、上皮细胞、成纤维细胞、神经元、星形胶质细胞等,外泌体几乎存在于所有体液如脑脊液、尿液、精子、唾液、血液、玻璃体和乳汁等[4],含有丰富的核酸、蛋白、脂质和代谢物等。外泌体所携带的物质由于源细胞的类型及其所处状态(例如转化、分化、刺激和压力)不同而存在很大差异,由其大小、内容物含量、对受体细胞的功能影响以及细胞来源来概念化,因此是一个高度异质性的群体,具有独特的诱导生物学反应的能力,也为一些疾病如代谢性疾病、心血管疾病、神经退行性疾病、肿瘤等提供诊断和预后信息[5]。此外,作为一种纳米粒径的内源性囊泡,高生物相容性、天然的归巢性能、以及进行功能化修饰后实现更特异性的组织器官和病灶部位的富集,都使得外泌体作为一种新型药物递送载体具有很好的研究价值。本文就外泌体的提取分离方法,在疾病中的诊疗作用以及工程化修饰后用于药物递送做一综述。
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外泌体的发生机制主要涉及质膜的双重内陷。质膜的第一次内陷形成早期分选内含体(ESEs),此时ESEs膜内表面上附着细胞表面蛋白和细胞外环境相关的可溶性蛋白,有时新形成的ESEs可直接与预先存在的ESEs合并。ESEs在高尔基体和内质网的帮助下形成晚期分选内含体(LSEs)并再次内陷形成多泡体(MVBs),MVBs包含数个腔内小泡(ILVs)。MVBs可以与溶酶体或自噬体融合以被降解,也可以与质膜融合以释放包含的ILVs,ILVs释放后即形成直径约40~160 nm的外泌体[6-7]。
目前常用的外泌体分离方法有超速离心法、密度梯度离心法、超滤法、色谱法、免疫亲和法、聚合物沉淀法等,分别采用不同的分离机制,所得到的外泌体具有不同的得率和纯度,其主要原理和优缺点见表1。
方法 原理 优势 耗时 纯度 产率 不足 超速离心法[8] 大小和密度不同的组分具有不同的沉积速度 金标准,适用于大批量样品,技术成熟 >4 h 中 低 仪器昂贵、操作繁琐耗时、产量低,可能会破坏外泌体[9] 密度梯度离心法 大小和密度不同的组分具有不同的沉积速度 高纯度,避免外泌体损伤 >16 h 高 低 前期准备、操作繁琐、耗时[10] 超滤法[11] 不同粒子粒径和相对分子质量的差异 操作简便,不需要特殊设备和试剂 <4 h 高 中 滤膜易堵塞,小粒径外泌体
易丢失[12]色谱法 不同粒子粒径和相对分子质量的差异 简单、经济,能较好保持外泌体生物功能和结构[13] <0.3 h 高 高 需要特殊的柱子和填料,存在脂蛋白污染 免疫亲和法[14] 抗体与外泌体特异性膜蛋白的相互作用 特异性分离外泌体 4~20 h 高 中 昂贵,耗时,分离效果取决于抗体的特异性 聚合物沉淀法[15] 外泌体在高亲水性聚合物影响下溶解度或分散性的变化 操作简单,适用于大体积
样品0.3~12 h 低 高 潜在污染物(提纯蛋白质聚集体或残留聚合物)
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