应用PEG 6000探讨滑膜组织外泌体的提取与鉴定

目的:应用PEG 6000提取滑膜组织中外泌体并进行鉴定。方法:收集于北京中医药大学第三附属医院骨科行膝关节镜或关节置换手术过程中废弃的滑膜组织,用8%PEG 6000溶液对滑膜组织分泌的外泌体进行富集、提取,应用透射电镜(TEM)、纳米颗粒跟踪分析(NTA)、Western Blot对外泌体的形态、粒径大小以及标志蛋白CD9、CD63和Flotillin-1进行检测。结果:应用PEG 6000溶液可成功提取出富集类圆形、盘状囊泡的提取物,NTA鉴定显示粒径为(98.6±10.8)nm,Western Blot显示外泌体标志蛋白CD9、CD63和Flotillin-1均呈阳性高表达。结论:通过对提取物的形态、粒径大小以及标志蛋白分析证实PEG 6000从滑膜组织提取到的物质为外泌体,为以后研究滑膜组织外泌体的功能和机制提供了方法学基础。

Research of extraction and identification of synovial tissues-derived exosomes using PEG 6000

Objective:The PEG 6000 was used to extract the synovial tissue-derived exosomes and then identified.Methods:The synovial tissue discarded during knee arthroscopy and joint replacement surgery was collected from the Department of Orthopedics,Beijing University of Chinese Medicine Third Affliated Hospital.The synovial tissue-derived exosomes were enriched and extracted with 8%PEG 6000 solution.Transmission Electron Microscopy(TEM),Nanoparticle Tracer Analysis(NTA),and Western Blot were used to detect the morphology,particle size and biomarker proteins(CD9,CD63,and Flotillin-1)of exosomes.Results:The extracts of enriched round and discoid vesicles were successfully extracted with PEG 6000 solution.The NTA identification showed a particle size of about(98.6±10.8)nm.Western Blot showed exosome marker proteins CD9,CD63,and Flotillin-1 were both positive and high expression.Conclusion:The substance extracted from the synovial tissue of PEG 6000 is exosome by the detection of morphology,particle size and marker protein,which provides a methodological basis for studying the function and mechanism of synovial tissue exosomes in the future.