豆状囊尾蚴外泌体的分离鉴定及其对家兔免疫应答的调节作用  

Isolation and characterization of exosomes derived from Cysticercus pisiformis and its immunomodulation on rabbits’immune response

作　　者：王立群 李艳萍 张少华[1] 梁盼红 李涛善 刘婷丽 骆学农[1,3] WANG Li-qun;LI Yan-ping;ZHANG Shao-hua;LIANG Pan-hong;LI Tao-shan;LIU Ting-li;LUO Xue-nong(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses/Yangzhou University,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2020年第3期307-313,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017FYD0501303);中央级公益性科研院所基本科研业务费专项(1610312020017);国家自然科学基金项目(31372433)。

摘　　要：为探究豆状囊尾蚴外泌体对家兔免疫应答的调节作用,以收集的豆状囊尾蚴分泌排泄产物(ESP)为材料,通过差速离心法分离外泌体,经透射电镜和Western-blot分别对其形态和标志分子进行鉴定。分别用外泌体和ESP免疫家兔,ELISA方法检测家兔血清中特异性抗体和血清细胞因子水平,并通过人工攻虫感染评价其免疫保护效果。结果显示,豆状囊尾蚴外泌体直径为30~150 nm,呈圆形或椭圆形,有完整的脂质双分子层膜结构,且含有外泌体标志蛋白CD63。而且,外泌体免疫组血清特异性抗体、IL-10和IL-4的水平显著升高(P<0.05),而IFN-γ和TNF-α显著降低(P<0.05)或差异不显著。经剖检发现,豆状囊尾蚴ESP免疫组和外泌体免疫组的减虫率分别为65.17%和24.88%。结果表明,外泌体通过上调宿主抑炎因子的表达,诱导宿主产生了Th2型细胞免疫应答,从而有利于囊尾蚴在宿主体内的存活。To explore the regulatory role of Cysticercus pisiformis exosomes in immune response to rabbits,the exosomes from excretory-secretory products(ESP)of C.pisiformis were isolated by differential centrifugation.The morphology of exosomes was observed by transmission electron microscopy(TEM)and the marker protein CD63 was analyzed by Western-blot.Subsequently,rabbits were immunized with exosomes and ESP respectively,and then the level of specific antibodies and cytokines were detected by ELISA.Moreover,the immunoprotective effect was evaluated after artificial challenge infection with T.pisiformis.The results showed that the C.pisiformis exosomes were 30 to 150 nm in diameter with a complete lipid bilayer membrane structure,which was spherical or oval,and reacted with anti-CD63 antibodies.The levels of specific antibodies,IL-10 and IL-4 were significantly increased in exosomes immunized rabbits(P<0.05),while the level of IFN-γwas significantly decreased(P<0.05)and TNF-αperformed no difference.The worm reduction rates for ESP group and exosomes group were 65.17% and 24.88% after necropsy,respectively.These results implied that exosomes of C.pisiformis could induce the Th2 type cellular immune response by promoting the host anti-inflammatory cytokines expression,which would contribute to the survival of Cysticercus in rabbits.

关 键 词：豆状囊尾蚴 外泌体 免疫调节 细胞因子 减虫率 

分 类 号：S852.734[农业科学—基础兽医学]