植物细胞外囊泡及其分析技术的进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-1106

摘要/Abstract

摘要：

细胞外囊泡是细胞在生理和病理条件下通过胞吐作用释放的具有磷脂双分子层结构的纳米级囊泡。细胞外囊泡作为蛋白质、核酸、脂质和代谢物等物质的载体，能够在细胞与细胞之间穿梭，行使物质传递、信息交流的功能，是细胞间通讯的重要媒介。近年来，植物中细胞外囊泡的研究也不断深入，其研究和分析技术也取得了很大进展。本文介绍了细胞外囊泡的组成，综述了植物中细胞外囊泡的生物学功能，分析了细胞外囊泡分离与富集方法的优缺点，以及原位成像技术的应用，最后对植物细胞外囊泡研究技术发展的重点进行了展望。

关键词: 细胞外囊泡, 功能, 分离方法, 原位检测

Abstract:

Extracellular vesicles（EVs）are nanoscale vesicles with a phospholipid bilayer structure released by cells through exocytosis under physiological and pathological conditions. EVs, as carriers of information such as proteins, nucleic acids, lipids, and metabolites, can shuttle between cells to play function of material transfer and information exchange, thus they are important for intercellular communication. In recent years, the research of EVs in plant has also been deepening, and there have been great progress in their research and analysis technologies. In this review, we briefly introduce the composition of extracellular vesicles. Then we summarize the current progress in the biological functions of EVs in plants. Furthermore, we analyze the advantages and disadvantages of EV isolation and enrichment methods, as well as the application of EVs in situ imaging technology. Finally, we prospect the potential technologies for the research of plant EVs.

Key words: extracellular vesicles, function, isolation method, in situ detection

张雪萍, 鲁雨晴, 张月倩, 李晓娟. 植物细胞外囊泡及其分析技术的进展[J]. 生物技术通报, 2023, 39(5): 32-43.

ZHANG Xue-ping, LU Yu-qing, ZHANG Yue-qian, LI Xiao-juan. Advances in Plant Extracellular Vesicles and Analysis Techniques[J]. Biotechnology Bulletin, 2023, 39(5): 32-43.

图/表 2

图1 胞外囊泡的产生和传递 A：哺乳动物EVs生物发生途经。（a）微囊泡途径：由细胞质膜直接出芽脱落并释放到细胞外。（b）外泌体途径：细胞质膜向内凹陷后发生内吞作用，形成早期内涵体。早期内涵体将进一步向内凹陷形成晚期内涵体，包被部分细胞质和某些物质（如核酸、高尔基体和细胞核的蛋白质），从而产生腔内囊泡（ILV），具有ILV的多泡体（MVB）会与细胞膜融合，其内容物（ILV）可释放到细胞外环境中。释放的囊泡被称为外泌体。（c）凋亡小体以凋亡细胞的小泡形式释放。这些EVs可以与细胞外基质相互作用，并将其中的内容物输送到靶细胞。参照Chen等[35]修改。B：植物EVs的生物发生途经。（a）EXPO途经：EXPO是一种球形双膜结构，能与质膜融合并释放单层膜囊泡；（b）Vacuole途径：植物细胞中含有水解酶和防御成分的液泡可以与质膜融合，并将防御物质释放到细胞外空间，抑制病原体的增殖。（c）MVB途径：植物中的MVB可以与质膜融合，然后将EVs释放到细胞外空间。参照Cong等[33]修改

Fig. 1 Formation and delivery of extracellular vesicle A: The mammalian EVs biogenesis pathway.（a）Microvesicle pathway: Direct budding from the cytoplasmic membrane and release outside the cell.（b）Exosome pathway: Endocytosis occurs after the inward depression of the cytoplasmic membrane to form early endosomes. Early endosomes will be further recessed inward to form late endosomes, which coat part of the cytoplasm and certain substances（such as nucleic acids, proteins in the Golgi apparatus, and nucleus）, resulting in intraluminal vesicles（ILVs）. Multivesicular bodies（MVB）with ILVs fuse with cell membranes, and their contents（ILVs）can be released into the extracellular environment. The released vesicles are called exosomes.（c）Apoptotic bodies are released as vesicles of apoptotic cells. These EVs can interact with the extracellular matrix and transport its contents to target cells. Refer to Chen et al[35]. B: The biogenesis pathway of plant EVs.（a）exocyst-positive organelle（EXPO）pathway: EXPO is a spherical double-membrane structure, which can fuse with plasma membrane to release monolayer vesicles;（b）Vacuole pathway: The vacuole containing hydrolases and defense components in plant cells can fuse with the plasma membrane and release the defense material into the extracellular space to inhibit pathogen proliferation.（c）MVB pathway: In plants, MVB can fuse with plasma membrane and release EVs into the extracellular space. Refer to Cong et al[33]

表1 主要EVs的分离方法及优缺点

Table 1 Isolation methods and advantages and disadvantages of main EVs

技术名称 Approaches	原理 Mechanism of enrichment	优点 Advantages	缺点 Disadvantages	参考文献 References
超速离心法 Ultracentrifugation	密度	“金标准”，操作简便，可以分离大量样品	持续时间长，设备昂贵，离心速度过高可能会影响EVs的完整性	[54⇓-56]
蔗糖密度梯度离心 Sucrose density gradient ultracentrifugation	密度	分离纯度高，没有其他化学品污染	持续时间长（>4 h），样本量大，需要超离心，回收率低	[48,57]
沉淀法 Coprecipitation	表面电荷	操作简单、快速、成本低，对EVs损伤小	分离的外泌体纯度通常很低	[58-59]
尺寸排阻色谱法 Size-exclusive chromatography	大小和分子量	高效、操作简单且耗时短，提取外泌体得率与纯度高，能保持EV的完整性	费用高，需要专用设备，色谱柱使用次数与加样体积有限制，复杂性大蛋白聚集体和脂蛋白与共分离	[60⇓-62]
超滤法 Ultrafiltration	大小	程序简单，允许同时处理多个样本，省时，可较好地保持EV活性	无法滤除小于滤膜孔径的杂质，样品使用前需去除细胞等大的膜状结构	[63-64]
微流控技术 Microfluidics technology	大小、密度和表面抗原亲和	小型化、集成化、高通量和耗费时间短	研究技术尚未标准化；成本高，设备昂贵	[35,65 -66]
免疫亲和捕获技术 Immunoaffinity capture-based technique	表面抗原亲和	高纯度，高特异性	抗体制备存在限制，成本高	[53,67 -68]

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