不同预处理及分离方法提取山羊血浆外泌体效果的研究  

Effects of different pretreatment and separation methods on extraction ofgoat plasma exosomes

作　　者：毛敏 林雅婷 伍翠莹 李耀坤[1] 刘德武[1] 柳广斌[1] MAO Min;LIN Yating;WU Cuiying;LI Yaokun;LIU Dewu;LIU Guangbin(College of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院,广州510642

出　　处：《黑龙江畜牧兽医》2023年第19期62-65,139,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省自然科学基金项目(2021A1515010636);广东省现代农业产业技术体系项目(20022KJ127);韶关市仁化县牛羊产业园山羊养殖技术服务项目(h20220665)。

摘　　要：为了探讨不同预处理及分离方法提取山羊血浆外泌体的效果,试验利用过滤膜对采集的血浆样品进行预处理后分为0.22μm过滤组、0.45μm过滤组和0.80μm过滤组,同时设置未过滤组,然后采用超速离心法和试剂盒法提取不同预处理组的血浆外泌体,通过透射电子显微镜、纳米粒径分析仪及Western-blot方法对血浆外泌体的形态、大小及标志蛋白的表达进行分析。结果表明:超速离心法和试剂盒法提取的血浆外泌体的粒径大部分分布在30~200 nm之间;外泌体的结构完整,大小不一,呈圆盘状或茶托样,并且0.22μm过滤组采用超速离心法提取的外泌体在透射电子显微镜下的形态最清晰;0.80μm过滤组采用试剂盒法提取的血浆外泌体纳米粒径显著大于超速离心法(P<0.05),未过滤组及不同过滤组采用试剂盒法提取的血浆外泌体蛋白浓度显著高于超速离心法(P<0.05);不同预处理及分离方法提取的血浆外泌体表面均有标志蛋白TSG101、CD63、CD9的表达。说明经0.22μm过滤膜预处理并结合超速离心法提取的血浆外泌体纯度最高,经0.80μm过滤膜预处理并结合试剂盒法提取的血浆外泌体产量最高。In order to investigate the effects of different pretreatment and isolation methods on the extraction of goat plasma exosomes,the collected plasma samples were pretreated with filter membranes and were divided into unfiltered group,0.22μm filtration group,0.45μm filtration group,0.80μm filtration group.Then,plasma exosomes from different pretreatment groups were extracted by ultracentrifugation method and kit method,and the morphology,size and expression of marker proteins of plasma exosomes were analyzed by transmission electron microscopy,nanoparticle size tracking analysis and Western-blot.The results showed that the particle size of most plasma exosomes extracted by both ultracentrifugation and kit methods were distributed in the range of 30-200 nm.The exosomes were complete in structure and varied in size.They were disc-like or saucer-like,and the exosomes extracted by ultrafiltration method in the 0.22μm filtration group showed the clearest morphology under transmission electron microscopy.The particle size of plasma exosomes extracted by the kit method was significantly higher than that by the ultracentrifugation method in the 0.80μm filtration group(P<0.05).The concentration of plasma exosomal proteins extracted by kit method was significantly higher in the unfiltered and differently filtered group than in the ultracentrifugation method(P<0.05).The concentration of exosome protein extracted by kit method was higher in different filter groups.TSG101,CD63 and CD9 were expressed on the surface of plasma exosomes extracted by different pretreatment and isolation methods.The results showed that the purity of plasma exosomes extracted by 0.22μm iltration membrane pretreatment combined with the ultracentrifugation method was the highest,and the yield of plasma exosomes extracted by 0.80μm filtration membrane pretreatment combined with the kit method was the highest.

关 键 词：山羊 血浆外泌体 过滤 超速离心法 试剂盒法 

分 类 号：S826[农业科学—畜牧学]