摘要/Abstract

外泌体是细胞主动分泌的一种纳米级双层膜结构的小囊泡，能够直接反映分泌细胞的生理和功能状态，进行细胞间的物质运输和信息通讯，并参与多种生理及病理过程.本文针对细胞分泌外泌体的动态过程，以DNA四面体为基础，结合细胞膜修饰技术和荧光成像技术，构建了一种易操作、高稳定性的细胞膜表面DNA四面体传感器，应用于不同细胞类型分泌外泌体的实时监测.DNA四面体传感器通过脚支链与疏水性胆固醇探针的杂交互补作用锚定于细胞膜表面，利用四跨膜蛋白CD63核酸适配体序列特异性捕获细胞膜表面释放的外泌体，通过监测细胞膜表面荧光的变化，实时测定外泌体的分泌情况.
关键词: 细胞膜表面传感器, DNA四面体, 外泌体, 分泌, 实时监测, 核酸适配体
Exosomes are nanoscale bilayer membrane vesicles actively secreted by cells, which carry abundant cell-specific substances. They can directly reflect the physiological and functional status of the secreting cells and play important roles in intercellular communication, physiological and pathological processes. In this work, we combined membrane modification technique with fluorescence imaging technique and blended CD63 aptamers into a highly stable and universal DNA tetrahedral structure to construct a cell membrane-anchored DNA sensor for real-time monitoring the secretion of exosomes. We designed four functional toes on each vertex of the tetrahedral sensor, respectively. A signal report toe on the top vertex consisted of fluorophore-modified CD63 aptamer, quencher-modified quencher probe(QP) binding part of the CD63 aptamer, and block probe (BP) binding the rest of the CD63 aptamer. The other three extended toes on the vertices were immobilized to the cell membrane by hybridizing with cholesterol-modified anchor probes(AP), which spontaneously incorporated to a lipid bilayer via hydrophobic interaction between the cholesterol moieties and the cellular membrane. In the initial state, the proposed DNA tetrahedral sensor was tethered to membrane with fluorophores quenched by QP and CD63 aptamer blocked by QP and BP. Trigger probes (TP) were add to bind to BP, resulting in the activation of the sensor. Subsequently, CD63 aptamers were specifically bound to the secreted exosomes, leading to the release of QP and concurrent fluorescence restoration of fluorophore. The intensity of the fluorescent signal in cell membrane was proportional to the amount of exosomes captured, thus realizing the real-time monitoring of the exosomes by analysis the changes of the fluorescence intensity. The experimental results showed that the sensor exhibited a good stability and a high capture efficiency for secreted exosomes. This strategy would provide a potentially useful tool for a variety of applications in biomedical research, drug discovery and tissue engineering.
Key words: cell membrane-anchored sensor, DNA tetrahedron, exosome, secretion, real-time monitoring, aptamer


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