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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。
表 1 外泌体的分离方法及特点
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)
Progress on exosomes in the diagnosis and treatment of disease and drug delivery system
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摘要: 外泌体是细胞外囊泡的一种,作为特殊的细胞间通讯介质,携带蛋白质、核酸及脂质等,在生物体内各种生理、病理过程中发挥着重要作用。作为内源性纳米囊泡,外泌体具有体循环稳定性、良好的生物相容性、对组织和细胞的特异性靶向等优点,是理想的药物递送载体。外泌体为多种疾病的诊断和预后评估提供支持,同时作为一种非常有潜力的、安全、特异性强的内源性纳米药物载体具有广阔的应用前景。本文阐述外泌体的产生机制,对其提取分离方法特点进行总结,并围绕外泌体在免疫和炎症相关疾病、心血管系统疾病、神经系统疾病、肿瘤等疾病的应用机制进行讨论,以及作为药物载体的工程化修饰和主动靶向药物递送进行综述。Abstract: As a type of extracellular vesicles, exosomes are released by living cells and contain diverse bioactive molecules, including nucleic acids, proteins, lipids and metabolites. They play an important role in various physiological and pathological processes by a special intercellular communication medium. As endogenous vesicles, exosomes also have the advantages of systemic circulation stability, good biocompatibility and specific targeting of tissues and cells, as well as they are promising candidates for drug delivery system. The production mechanism of exosomes describe was summarized, the methods of extraction and separation the application and mechanism of exosomes in immune and inflammation-related diseases, cardiovascular system diseases, nervous system diseases, tumors, etc. were reviewed. The engineering modifications of exosomes in high targeting properties based on the drug delivery were overviewed. Exosomes support the diagnosis and prognostic assessment of multiple diseases, which have broad application prospects as a very potential safe and specific endogenous nano-drug carrier.
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Key words:
- exosome /
- targeted modification /
- targeted therapy /
- drug delivery
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表 1 外泌体的分离方法及特点
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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