关键词: 外泌体, 超速离心法, 膜亲和法, 沉淀法

Abstract:

Objective To compare the efficiency of ultracentrifugation,affinity membrane method and precipitation for extracting plasma exosome. Methods Plasma exosomes were extracted by 3 different methods,including ultracentrifugation,affinity membrane method and precipitation. Transmission electron microscopy,nano-flow cytometry,zetasizer nano were used to verify its morphology,particle size and distribution and Zeta potential. The western blot analysis was performed to measure the representative protein markers of exosomes. The bicinchoninic acid assay was utilized to measure concentration of exosomal protein. The ratio of particles number to protein concentration was analyzed to evaluate the extraction efficiency of exosomes. Results The transmission electron microscope showed exosomes extracted by ultracentrifugation and affinity membrane method as a saucer-like bilayer membrane structure,while the exosomes extracted by precipitation were mixed with PEG polymer and protein particles. The size of the exosomes were between 40-100 nm,while the mean diameter of the exosomes derived using affinity membrane method was larger than those using the ultracentrifugation and precipitation. Dynamic light scattering（DLS） analysis showed that the Zeta potential of exosomes was negative,which implied their membranous structure. However,exosomes size measured by DLS were larger than that by nano-flow cytometry. In addition,all the exosomes expressed representative protein markers. The precipitation produced the highest protein concentration and particles,and the ultracentrifugation gave exosomes the highest purity than the other 2 methods. Conclusions Exosomes can be extracted by ultracentrifugation,affinity membrane and precipitation. However,which extraction method should be used depends on the specific experimental requirements.

Key words: Exosomes, Ultracentrifugation, Affinity membrane method, Precipitation