旋毛虫肌幼虫期胞外囊泡对小鼠免疫保护作用  

Immune protection effects of Trichinella spiralis muscle larvae extracellular vesicles on mice

作　　者：徐枫雁 张媛媛 刘晓雷 刘明远 王学林 王洋 白雪 XU Fengyan;ZHANG Yuanyuan;LIU Xiaolei;LIU Mingyuan;WANG Xuelin;WANG Yang;BAI Xue(Key Laboratory of Zoonosis Research of Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2022年第2期277-284,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFC1601200,2018YFC1602500)。

摘　　要：本试验通过差速超速离心法获得旋毛虫肌幼虫期胞外囊泡(Trichinella spiralis muscle larvae extracellular vesicles,Ts-ML-EVs),经透射电镜观察、纳米颗粒追踪分析、流式细胞术和Western blot鉴定。选择6~8周龄的健康雌性BALB/c小鼠,随机分为4组,每组8只。试验设计为PBS对照组(PBS组)、佐剂对照组(PBS+佐剂组)、旋毛虫肌幼虫期排泄分泌产物免疫组(Ts-ML-ES+佐剂组)以及旋毛虫肌幼虫期胞外囊泡免疫组(Ts-ML-EVs+佐剂组),分别取相应抗原与佐剂等体积混合采用多点皮下注射方式免疫小鼠。首免后第2,4周各加强免疫1次,剂量不变。三免后2周,每只小鼠灌胃300条旋毛虫肌幼虫,灌胃后6 d每组取2只剖杀,统计旋毛虫成虫减虫结果;攻虫35 d后所有小鼠全部处死,计算肌幼虫减虫率。每次免疫前、攻虫前以及处死前眼眶采血收集血清并保存,统一进行ELISA分析。结果显示:成功获得Ts-ML-EVs,随着免疫次数增加,小鼠血清中特异性IgG、IgG1、IgG2a、IgA和IgE抗体水平均显著上升,在三免后2周达到最高;而IgM在二免后2周抗体水平达到最高,三免后抗体水平开始下降。Th1(IFN-γ、IL-12)和Th2(IL-4、IL-10)型细胞因子水平同样显著升高,在三免后2周达到最高,且Th2型变化更加明显,表明Ts-ML-EVs可以使小鼠产生体液免疫和以Th2型免疫反应为主的细胞免疫应答。Ts-ML-EVs的成虫和肌幼虫减虫率分别为23.4%和43.7%,表明Ts-ML-EVs对小鼠具有较好的免疫保护效果,试验为研制旋毛虫病新型高效疫苗奠定基础。In this study, the Trichinella spiralis muscle larvae extracellular vesicles(Ts-ML-EVs) were obtained by ultracentrifugation.Bilayer vesicles were identified by observation under transmission electron microscopy, nanoparticle tracking analysis, flow cytometry analysis and Western blot.Thirty-two aged 6 to 8 weeks healthy mice were chose and divided into 4 groups: PBS control group(PBS group),adjuvant control group(PBS + adjuvant group),Trichinella spiralis muscle larvae excretory secretory immunize group(Ts-ML-ES + adjuvant group) and Trichinella spiralis muscle larvae extracellular vesicles group(Ts-ML-EVs + adjuvant group).Mice were vaccinated subcutaneously with mixed antigen + adjuvant two and four weeks after first vaccination.The immunization was strengthened once at two and four weeks after the first immunization, and the dose remained unchanged.Two weeks after the third immunization, each mouse was gavaged with 300 Trichinella spiralis muscle larvae, 6 d later, 2 mice from each group were chose to count adult worm burden, then thirty-five days after infection, all the mice were euthanasia, then larvae burden was count.Before each immunization,infection with muscle larvae and euthanasia,the serum was collected to carry out the next experiment.The results showed that Ts-ML-EVs were obtained successfully,the specific IgG,IgG1,IgG2 a,IgA and IgE antibody levels in serum were significantly increased with the increase of immunization times,and reached the highest level two weeks after the third immunization,while the IgM antibody level reached the highest level two weeks after the second immunization,and the antibody level began to decline after the third immunization.The levels of Th1(IFN-γ,IL-12)and Th2(IL-4,IL-10)cytokines were also significantly increased,and reached the highest level two weeks after the third immunization,and the changes of Th2 type were more obvious,suggesting that Ts-ML-EVs could induce humoral immunity and cell-mediated immunity response mainly based on Th2 type immune response in mice

关 键 词：旋毛虫 胞外囊泡 小鼠 免疫保护 

分 类 号：S852.73[农业科学—基础兽医学] R535[农业科学—兽医学]