保存时间对血浆胞外囊泡及囊泡蛋白的影响

Effects of storage time on plasma extracellular vesicles and their proteins

目的:探讨不同保存时间对血浆胞外囊泡(extracellular vesicles,EVs)及肿瘤相关蛋白PD-L1表达的影响。方法:使用离心和免疫吸附的方法,选取回顾性研究常用的-80℃存放1个月、1年、3年、5年的血浆EVs与新鲜血浆EVs进行对比;使用透射电镜对提取的EVs的形态进行观察;用NTA检测各组EVs的大小及数量;用ELISA试剂盒测定EVs上肿瘤标志性蛋白PD-L1的表达情况。结果:不同存储时间的血浆总蛋白水平及平均粒径大小无显著差异。而保存时间超过1年,EVs形态出现变形、聚集和碎裂等变化。随着保存时间的延长,EVs上的标志性蛋白CD63逐年减少。保存3年和5年的样本中肿瘤标志蛋白PD-L1水平明显降低。结论:存储时间对EVs表面蛋白及肿瘤标志蛋白影响较大。在以EVs为对象的研究中,需尽量采用随访设计,提取新鲜的血浆样本进行检测。在回溯性研究中,尽量选取样本库中保存一年以内的样本进行检测。

Objective:To investigate the effects of different storage times on the expression of plasma extracellular vesicles( EVs) and tumor-associated protein PD-L1.Methods:EVs in plasma stored at-80 ℃ for 1 month,1year,3 years and 5 years were compared with EVs in fresh plasma by centrifugation and immunosorption.The morphology of the extracted EVs was observed by transmission electron microscopy.The size and quantity of EVs in each group were detected by NTA.The expression of PD-L1 on EVs was determined by ELISA kit.Results:There was no significant difference in plasma total protein level and average particle size for different storage times.When the EVs were stored for more than 1 year,the morphology of EVs changed,such as deformation,aggregation and fragmentation.With the extension of storage time,the signature protein CD63 on EVs decreased year by year.Levels of the tumor marker protein PD-L1 were significantly reduced in samples stored for 3 and 5 years.Conclusion:Storage time has a significant effect on EVs surface protein and tumor marker protein.In the study of EVs,follow-up design should be adopted as far as possible,and fresh plasma samples should be extracted for detection.In the retrospective study,samples stored within one year in the sample bank were selected as far as possible for detection.