基于干涉等离子体显微成像技术的外泌体亚型研究

Exosomes are membrane-vesicles released by cells. They contain numerous bioactive molecules, and play an important role in tumorigenesis, tumor growth, metastasis and tumor cell resistance. The technique for exosome detection and analysis is one of the hottest topics in cancer diagnosis and targeted drug delivery. Exosomes are composed of vesicle population with a diameter of 30 to 120nm. It is more accurate to treat it as several subsets, as it will benefit our understanding of their biogenesis, molecular compositions and functions. However, separation of different subsets of exosomes is extremely challenging because of their tiny size, so there is not much research based on the current detecting techonology that is focused on their heterogeneity. Here we proposed to apply interferometric plasmonic microscopy to the analysis of distribution and membrane proteins of different exosome subsets without complicated separation process. IPM is a kind of label-free imaging technology based on the surface plasmon microscopy, which can image single exosomes and obtain the physical (size) and biochemical information (interaction with antibody) of exosomes at the same time. Exosome subset analysis based on IPM does not require the complicated subset separation and sample preparation. IPM provides a much easy and efficient approach for exosome subset analysis, and promotes the basic research and clinical applications of exosomes.

外泌体是由细胞分泌的膜性囊泡，含有多种生物活性物质，在肿瘤的发生、生长、转移以及抗肿瘤免疫等诸多方面起着重要的作用。外泌体粒径分布在30-120nm之间，根据粒径将其细分为不同亚型，可以更准确地对外泌体的产生、分子组成和功能进行研究。但是由于外泌体非常微小，准确细分不同粒径亚型非常困难，所以目前大部分检测方法无法对其这一异质性特点进行研究。本研究拟将干涉等离子体显微成像（iPM）技术应用于外泌体亚型分析中，实现无需外泌体亚型分离，直接对不同亚型外泌体膜蛋白组成进行分析。iPM是一种基于表面等离子体共振的免标记成像技术，具有外泌体单颗粒成像的能力，而且能够同时获取外泌体物理（粒径）和生物化学（抗体特异性吸附）信息。基于iPM的外泌体亚型分析，无需复杂的外泌体亚型样本的分离和制备，为外泌体亚型分析提供了一种简单而高效的分析手段，有助于推进外泌体的基础研究和临床应用。

外泌体是由细胞分泌的膜性囊泡，含有多种生物活性物质，在肿瘤的发生、生长、转移以及抗肿瘤免疫等诸多方面起着重要的作用。外泌体粒径分布在30-120nm之间，根据粒径将其细分为不同亚型，可以更准确地对外泌体的产生、分子组成和功能进行研究。但是由于外泌体非常微小，准确细分不同粒径亚型非常困难，所以目前的检测方法无法对其这一异质性特点进行研究。本研究开发了基于干涉等离子体显微成像（iPM）技术的外泌体亚型分析方法，利用其原位、动态成像能力，突破了外泌体异质性分析的瓶颈，实现了无需外泌体亚型分离，直接对不同亚型外泌体膜蛋白组成进行分析。研究中选用五种不同系细胞进行培养，分别为A549肺癌、HepG2肝癌、MCF-7乳腺癌、LNCaP前列腺癌、L-02正常细胞系，并用超速离心法进行了外泌体样品收集。其次，设计了多通道微流控系统，优化了芯片表面修饰流程，在此基础上进行了外泌体单颗粒与多种抗体的动态结合的实验，构建了不同粒径外泌体表面蛋白标志物信息矩阵，并进一步探索了基于外泌体亚型分析的外泌体来源诊断。实验结果不仅证明了iPM在肿瘤诊断方面的巨大应用潜力，更提供了直接证据证明在亚型尺度分析外泌体的必要性，为未来外泌体研究指明了方向。