两种提取细胞外泌体方法的比较

COMPARISON OF TWO METHODS TO EXTRACT CELLS EXOSOMES

近期研究发现外泌体在很多生理病理过程中起着重要作用,因而外泌体已成为当前的科研热点。本研究从外泌体形态、标志蛋白的表达方面比较了He La细胞外泌体的两种常用提取方法,以期获得更有效地提取Hela细胞中的外泌体方法。利用电镜观察两种方法提取的外泌体形态;动态光散射(dynamic light scattering,DLS)测定外泌体颗粒大小及分布,蛋白印迹法(Western Blot,WB)检测外泌体标志蛋白CD63、CD81的表达。结果显示,差速离心法与PEG沉淀法所提外泌体都呈茶托样双层膜结构,直径约30~150 nm,无明显形态差异,外泌体标志蛋白正常表达,但PEG沉淀法要引入PEG,有可能影响外泌体生物活性,可见,差速离心法是一种更可靠的有效提取外泌体的方法。

Recent studies have found that exosomes play an important role in many physiological and pathological processes.Thus,exosomes have become a current research hotspot.We compared two common methods of extracting HeLa exosomes to obtain some reliable and effective exosomes.The morphology of exosomes extracted by differential centrifugation method and polyethylene glycol(PEG)precipitation method was observed by electron microscopy.The size and distribution of particles from the samples was detected by dynamic light scattering(DLS).The expression of the exosome marker protein,CD63 and CD81,was analyzed by Western blot.We found that exosomes extracted with differential centrifugation method and PEG precipitation method both have saucer like membrane structure,and their diameters are around 30~150 nm.There are no signifi cant morphological differences between exosomes isolated with these two methods.The exosome marker proteins were normally expressed.Thus,compared with PEG precipitation method,differential centrifugation method could be a more reliable and effective method of extracting exosomes.