细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导小鼠脾细胞亚群的变化

目的 探讨细粒棘球绦虫(Eg)转Eg95-EgA31融合基因苜蓿疫苗免疫和Eg原头节攻击后小鼠脾CD4+和CD8+T淋巴细胞亚群的变化.方法 热絮凝法提取转基因苜蓿叶蛋白,用无菌双蒸水将叶蛋白提取液配成20 g/L,同时提取转空质粒(pBI121)苜蓿叶蛋白及正常苜蓿叶蛋白作对照.32只雌性Balb/c小鼠按体质量随机分为4组,每组8只.口服灌胃组:灌胃接种100 μl转基因苜蓿叶蛋白提取液;鼻腔黏膜接种组:滴鼻接种10 μl转基因苜蓿叶蛋白提取液;空质粒对照组:滴鼻接种10μl转空质粒苜蓿叶蛋白提取液;正常蛋白对照组:灌胃接种100μl正常苜蓿叶蛋白提取液.小鼠每3天免疫1次,连续免疫2个月.末次免疫后第8周,各组小鼠用Eg原头节腹腔注射攻击感染(50个/只),感染后第24周剖杀小鼠,分离脾细胞,用流式细胞仪检测脾CD4+和CD8+T淋巴细胞亚群百分比.结果 口服灌胃组小鼠脾CD4+T细胞亚群百分比(0.286±0.009)、CD8+ T细胞亚群百分比(0.102±0.004)和CD4+/CD8+比值(2.814±0.014)均显著高于正常蛋白对照组(0.166±0.018、0.083±0.006、2.019±0.369,P<0.01或<0.05);鼻腔黏膜接种组小鼠脾CD4+T细胞亚群百分比(0.269±0.016)和CD4+/CD8+比值(2.955±0.986)与正常蛋白对照组比较明显升高(P均<0.01);口服灌胃组小鼠脾CD4+T细胞亚群百分比高于鼻腔黏膜接种组(P<0.05);空质粒对照组小鼠脾CD4+、CD8+T细胞百分比和CD4+/CD8+比值(0.169±0.018、0.093±0.019、1.852±0.188)与正常蛋白对照组比较,差异无统计学意义(P均>0.05).结论 CD4+T细胞亚群可能与细粒棘球绦虫转Eg95-Eg31融合基因苜蓿疫苗诱导的小鼠抗Eg原头节攻击感染的保护力有关.疫苗灌胃接种可能是一种较好的免疫途径.

The change of splenocyte subsets in Balb/c mice by immunization with the transgenic alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus

Objective To investigate the change of splenocyte subsets in Balb/c mice immunized with transgenic alfalfa(Medicago sativa)containing Eg95-EgA31 fusion gene of Echinococcus granulosus(Eg) and challenged with Eg protoscoleces.Methods Leaf protein was extracted from transgenic alfalfa containing Eg95-EgA31 fusion gene by heat-coagulation method,and concentration of 20 g/L was used in the study.Meanwhile,leaf protein extracted from the transgenic alfalfa containing blank vector(pBI121)and the normal alfalfa was served as control.Thirty-two female Balb/c mice were randomly divided into 4 groups,8 mice in each group.Oral group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intragastrically(100μl per mouse);intranasal group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intranasally(10 μl per mouse);blank vector group was vaccinated intranasally with 10μl leaf protein with blank vector(pBI121);and normal control group was given 100μl normal leaf protein intragastrically.All mice in the above mentioned groups were immunized every 3 days for 2 months.Then,the mice were challenged intraperitoneally with Eg protoscoleces(50 protoscoleces per mouse)8 weeks after last vaccination and sacrified 24 weeks pest infection to separate the splenocytes.Flow cytometry was used to measure the percentages of CD4+ and CD8+ T ceils subsets.Resuits Compared with the normal control group(0.166±0.018,0.083±0.006,2.019 ±0.369),the percentages of CD4+(0.286±0.009)and CD8+(0.102±0.004)T cell subsets and the ratio of CD4+/CD8+(2.814±0.014)in oral group increased significantly (P<0.01 or<0.05).The percentage of CD4+ subset(0.269±0.016)and the ratio of CD4+/CD8+(2.955±0.986) in intranasal group was significantly higher than that ofthe normal control group(all P<0.01).The percentage of CD4+ subset in oral group was significantly higher than that of the intranasal group(P<0.05).No significant difference was found in the percentages of CD4+ and CD8+ T cell subsets and the ratio of CD4+/CD8+ between the blank vector group(0.169±0.018,0.093±0.019,1.852±0.188)and the normal control group(all P>0.05).Conclusions CD4+ T cell may play an important role in the protection induced by transgenic alfalfa vaccine against the challenge of Eg protoscoleces.Intragastrical immunization may be a good route.