壳聚糖-pJME/GM-CSF纳米DNA疫苗肌注BALB/c鼠诱导细胞免疫效应的研究  

Effects of Cell-Mediated Immunity Induced by Intramuscular Chitosan-pJME/GM-CSF Nano-DNA Vaccine in BALB/c Mice

作　　者：翟永贞[1] 周言[1] 马力[1] 冯国和[1] 

机构地区：[1]中国医科大学附属盛京医院感染科,沈阳市110004

出　　处：《病毒学报》2014年第4期423-428,共6页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(30471547);辽宁省自然科学基金资助项目(20042072);辽宁省博士科研启动基金项目计划(20131147)

摘　　要：探讨载有日本脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白与粒细胞-巨噬细胞集落刺激因子(Granulocyte-macrophage colony-stimulating factor,GM-CSF)编码基因重组子(命名为pJME/GM-CSF)的壳聚糖纳米颗粒的免疫佐剂效应及其机制。本研究采用复凝聚法制备壳聚糖-pJME/GM-CSF纳米颗粒;免疫组化法检测肌肉注射部位浸润细胞的类型,流式细胞仪检测不同免疫原免疫鼠后脾脏DC表型和功能的变化;乳酸脱氢酶释放法检测CTL活性。结果显示制备的壳聚糖-pJME/GM-CSF纳米颗粒鉴定正确;pJME/GM-CSF募集包括非成熟树突状细胞、巨噬细胞、粒细胞到注射部位,增加脾脏树突状细胞表面MHCII的表达,抗原摄取和递呈功能,增强疫苗诱导的细胞免疫反应,壳聚糖-pJME/GM-CSF纳米颗粒可进一步扩大pJME/GM-CSF的上述作用。这些结果表明壳聚糖可作为DNA疫苗肌注免疫时的传送载体,能够增强pJME/GM-CSF疫苗的细胞免疫应答。This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF.In this study,plasmid DNA（pJME/GM-CSF）was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process.Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection.The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly.The killing activity of CTLs was assessed using the lactate dehydrogenase（LDH）release assay.The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results.Our results also found that,after pJME/GM-CSF injection,the incoming cells were a mixture of macrophages,neutrophils,and immature DCs.Meanwhile,pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs,and enhanced their Ag capture and presentation functions.Cell-mediated immunity was induced by the vaccine.Furthermore,chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment,antigen processing and presentation,and vaccine enhancement.These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations,and enhance pJME/GM-CSF-induced cellular immune responses.

关 键 词：脑炎病毒 日本 壳聚糖 GM-CSF 树突状细胞 纳米颗粒 T淋巴细胞 细胞毒性T淋巴细胞 

分 类 号：R373.9[医药卫生—病原生物学]