细粒棘球绦虫亲肌肉抗原重组蛋白诱导小鼠免疫应答的研究

Study on Immune Response in ICR Mice by Immunization with Recombinant Myophilin Vaccine against Echinococcus granulosus

目的探讨细粒棘球绦虫(Echinococcus granulosus,Eg)亲肌肉抗原重组蛋白诱导ICR小鼠产生的免疫应答及其对Eg原头节攻击感染的保护性作用。方法 36只雄性ICR小鼠随机分为A(重组蛋白免疫组)、B(空质粒蛋白免疫组)和C(PBS对照组)等3组,每组12只。3组小鼠分别经皮下注射重组蛋白(10μg/只)、空质粒蛋白(10μg/只)和PBS(100μl/只),第2和4周同法加强免疫2次。末次免疫后2周,每只小鼠腹腔接种细粒棘球蚴原头节50个进行攻击感染。感染后20周,剖杀小鼠,分离并称重细粒棘球蚴组织,计算囊重减少率。取脾脏,分离脾细胞,流式细胞仪检测脾脏CD4+T和CD8+T淋巴细胞亚群百分比。脾细胞体外经脾细胞悬液、Eg粗抗原(EgAg)和伴刀豆球蛋白A(ConA)刺激培养4～5 h后,四甲基偶氮唑盐比色法(MTT法)检测T淋巴细胞增殖情况。结果感染后20周,A组小鼠包囊质量[(0.019±0.036)g]明显低于C组[(0.058±0.057)g](P<0.05),囊重减少率为69.1%;CD4+T和CD8+T细胞亚群百分比分别为(29.7±0.9)%和(9.7±0.8)%,均高于C组[(11.6±1.4)%和(7.8±0.2)%](P<0.01和P<0.05),CD4+/CD8+比值(3.061±0.015)也显著高于C组(1.487±0.106)(P<0.01)。未经刺激时,A组小鼠脾T淋巴细胞增殖水平(0.237±0.009)高于C组(0.159±0.005)(P<0.01);用EgAg和ConA刺激后,A组小鼠脾T淋巴细胞增殖水平[(0.283±0.008)和(0.325±0.025)]均高于C组[(0.203±0.002)和(0.244±0.006)](P<0.01)。结论棘球蚴亲肌肉抗原重组蛋白可诱导免疫小鼠脾脏T淋巴细胞增殖,CD4+T细胞亚群在重组蛋白诱导的小鼠抗Eg原头节攻击感染的保护性免疫机制中起一定作用。

Objective To investigate the immune response and the protection in mice induced by the recombi-nant myophilin protein of Echinococcus granulosus. Methods Thirty-six male ICR mice of 6-8 weeks old were randomly divided into groups A, B and C each with 12. The mice in the 3 groups were subcutaneously immunized with Eg myophilin protein, blank plasmid protein or PBS, respectively, by 3 times and challenged with protoscoleces of E. granulo-sus 2wk after the last vaccination. Mice were sacrificed 20wk after the infection, the hydatid cysts were collected for mea-suring the weight reduction. Spleens were obtained and the splenocytes were separated and cultured in vitro with EgAg or ConA stimulus for 4-5 h. The subsets of CD4＋ and CD8＋ T cells were measured by FACsort. The proliferation of spleno-cytes was determined by MTT method with blank plasmid and PBS as control. Results The average weight of the hy-datid cysts in the immunized group decreased by 69.1% in comparison to the blank plasmid and PBS groups. The CD4＋ subset [（29.7±0.9）%] and CD8＋ subset [（9.7±0.8）%] in group A increased significantly than group C, [（11.6±1.4）%] and [（7.8±0.2）%] respectively （P〈0.01 or 〈0.05）. The ratio of CD4＋/CD8＋ subsets in group A （3.061±0.015） was also higher than group C （1.487±0.106） （P〈0.01）. Without stimulation, the proliferation of T lymphocytes in group A（0.237±0.009）was high-er than group C （0.159±0.005） （P〈0.01）, with EgAg or ConA stimulus, it was also higher in group A than that of group C（P〈0.01）. Conclusion The recombinant myophilin protein of E. granulosus can induce the proliferation of splenocytes and Th1 response in mice, and the CD4＋ T cells subset may bear a part in the induced protection against the challenge of protoscoleces.