人脐血血浆外泌体提取方法的比较  

Comparison of the extraction methods of plasma exosomes from human umbilical cord blood

作　　者：叶俊杰 孙晓东 袁乐永[1,5] 冯胜蓝 覃冰清 许倩倩 谢丽霞 桑明 YE Junjie;SUN Xiaodong;YUAN Leyong;FENG Shenglan;QIN Bingqing;XU Qianqian;XIE Lixia;SANG Ming(Institute of Basic Medical Science,Hubei University of Medicine,Shiyan 442000,Hubei,China;Center for Translational Medicine,Xiangyang No.1 People’s Hospital,Hubei University of Medicine,Xiangyang 441000,Hubei,China;Hubei Clinical Research Cencer of Parkinson’s Disease,Xiangyang 441000,Hubei,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,Hubei,China;Southern University of Science and Technology Hospital,Shenzhen 518000,Guangdong,China)

机构地区：[1]湖北医药学院基础医学院,湖北十堰442000 [2]湖北医药学院附属襄阳市第一人民医院转化医学中心,湖北襄阳441000 [3]湖北省帕金森病临床研究中心,湖北襄阳441000 [4]湖北医药学院武当特色中药研究湖北省重点实验室,湖北十堰442000 [5]南方科技大学医院,广东深圳518000

出　　处：《生物工程学报》2022年第6期2342-2351,共10页Chinese Journal of Biotechnology

基　　金：湖北省对外科技合作项目(2019AHB068);湖北省实验动物资源开发及利用项目(2020DFE025);武当特色中药研究湖北省重点实验室(湖北医药学院)开放课题(WDCM2021011);湖北医药学院药护学院团队项目(2017YHKT02);襄阳市科技局医疗卫生领域科技计划重点项目(2020YL26)。

摘　　要：为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。We compared ultracentrifugation, sucrose gradient centrifugation, improved ultracentrifugation, and polyethylene glycol(PEG) precipitation in the extraction of plasma exosomes from human umbilical cord blood, aiming at screening out a stable and efficient method. The morphology, structure, and size of exosomes were observed based on transmission electron microscopy and dynamic light scattering. Total protein content of exosomes was determined by bicinchoninic acid(BCA) assay, and the expression of exosome markers CD63 and HSP70 and exosome negative marker GM130(Golgi marker) by Western blotting. Results showed that sucrose gradient centrifugation was more stable and yielded exosomes of uniform particle size compared with ultracentrifugation which had been considered as the “gold standard” for exosome extraction. However, sucrose gradient centrifugation had the limitations of complex operation and time-intensiveness. The improved ultracentrifugation featured ease of implementation and the extracted exosomes were of high purity.PEG precipitation extracted the most exosomes in a shorter timeframe, but the purity of the exosomes was low. In conclusion, all the four methods can separate exosomes from human umbilical cord blood plasma, but they are different in operation time, product purity, and product content. Therefore, the method for extracting plasma exosomes from human umbilical cord blood should be selected based on the experimental purpose and specific requirements.

关 键 词：外泌体 超高速离心法 蔗糖垫密度梯度离心法 改良超速离心法 聚乙二醇沉淀法 脐血血浆 

分 类 号：R714.5[医药卫生—妇产科学] R446.1[医药卫生—临床医学]