细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导Balb/c小鼠免疫应答的动态观察  

Dynamic observation on immune responses in Balb/c mice immunized with the transgenic alfalfa containing Eg95-EgA31 fusion gene of Echinococcus granulosus

作　　者：叶艳菊[1] 李文桂[1] 

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,400016

出　　处：《免疫学杂志》2010年第7期579-584,共6页Immunological Journal

基　　金：国家自然科学基金项目(30671835)

摘　　要：目的动态观察细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫Balb/c小鼠后诱导的免疫应答。方法热絮凝法提取转基因苜蓿的叶蛋白,再用无菌双蒸水将叶蛋白提取液的浓度配制成20μg/μl。88只Balb/c小鼠随机分为2组,分别用100μl灌胃和10μl滴鼻免疫小鼠,每3天1次,连续免疫2个月。在末次免疫后0、2、4、6、8、10、12、14、16、18和20周各组随机剖杀4只小鼠,眼球取血,常规酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清中IgG及其亚类和IgE水平;取脾,分离脾细胞,流式细胞仪(flow cytometr,FCM)检测脾CD4+和CD8+T淋巴细胞亚群的百分比,体外经脾细胞悬液或加入Eg粗抗原(EgAg)、伴刀豆球蛋白A(ConA)或脂多糖(LPS)刺激培养,四甲基偶氮唑盐比色法(MTT法)检测免疫鼠脾T淋巴细胞增殖情况,ELISA法检测脾细胞培养上清液中IL-12、IL-10、IFN-γ和TNF-α水平。结果在末次免疫后4～6周,2组免疫小鼠的血清IgG及其亚类和IgE水平升高,脾T淋巴细胞增殖水平升高,CD4+和CD8+T细胞亚群百分比分别升高,脾细胞培养上清液中IL-12、IFN-γ、TNF-α和IL-10水平分别升高。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗在免疫早期(4～6周)可诱导免疫鼠脾T淋巴细胞增殖,产生Th1和Th2混合型免疫应答,CD4+和CD8+T细胞亚群在细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导的保护性免疫机制中起重要作用。The aim of our study is to investigate the immune responses in Balb/c mice immunized by the transgenic alfalfa（Medicago sativa） containing Eg95-EgA31 fusion gene of Echinococcus granulosus（Eg）.We extracted the leaf protein from the trans genic alfalfa by heat-coagulation method,and adjusted its concentration to 20 μg/μl.Then,88 Balb/c mice were divided into 2 groups randomly,and immunized intranasally or orally with the leaf protein once per 3 days for 2 months.We sacrificed 4 mice from each group to collect the sera from the eyeballs on week 0,2,4,6,8,10,12,14,16,18,and 20 after the last immunization.By using ELISA,the levels of IgG,IgG subclasses,and IgE were evaluated.At the same time,splenocytes were separated and cultured in vitro with EgAg or ConA or LPS stimulus.The CD4＋ and CD8＋ T cell subsets were also measured by FCM.The splenocyte proliferation was determined by MTT method.The supernatant was collected for measuring the levels of IL-12,IL-10,IFN-γ and TNF-α by ELISA.We found that in week 4 to 6 after the last immunization,the splenocyte proliferation in the two immunized groups increased significantly and the CD4＋ and CD8＋ T cell subsets did so.Furthermore,the similar increasing trend about the levels of IL-12,IFN-γ,TNFα,and IL-10 as well as the antibody titers of IgG,IgG subclasses,and IgE could be seen in the two groups.We concluded that the proliferation of splenocytes and the mixed responses of Th1 and Th2 types can be induced by immunization with the transgenic alfalfa in the early period post immunization（4-6 weeks）.CD4＋ and CD8＋ subsets may play important roles in the protection induced by the transgenic alfalfa.

关 键 词：细粒棘球绦虫 转基因植物 苜蓿 细胞因子 脾T细胞亚群 

分 类 号：R392[医药卫生—免疫学]