弓形虫复合粘膜疫苗滴鼻免疫小鼠诱导的Peyer's patches持续性细胞免疫应答

Persistent cellular immune responses of Peyer's patches in mice induced by intranasal immunization with Toxoplasma gondii mucosal vaccine in mice

目的以可溶性速殖子抗原（soluble tachyzoite antigen，STAg）和霍乱毒素（choleratoxin，CT）佐剂制备的弓形虫复合粘膜疫苗滴鼻免疫小鼠，观察肠粘膜诱导部位Peyer’s patches（PP）的细胞免疫应答及持续时间，探讨其免疫机制。方法BALB／c小鼠96只，随机分为实验组和对照组，实验组以STAg（20μg／只）为抗原，CT（1／μg／只）为佐剂滴鼻免疫，对照组PBS滴鼻。滴鼻2次（间隔2周）后，每组6只小鼠分别于第1、2、3、4、6、8、10、12周处死。计数PP个数，制备PP淋巴细胞悬液，计数并涂片；免疫细胞化学法检测CD4^＋、CD8^＋T细胞亚群。结果实验期间两组小鼠PP数目均无明显变化；实验组免疫后PP淋巴细胞数量明显增生，第2周达高峰，第1、2、3周显著高于对照组（P〈0.05），其中以CD4^＋T细胞增生为主，第1周～第8周高于对照组（P〈0.01），CD8^＋T细胞第1周～第4周显著增高（P〈0.01），CD4^＋／CD8^＋比值无显著变化（P〉0．05）。结论弓形虫复合粘膜疫苗滴鼻免疫BALB／c小鼠可有效诱导肠PP部位持续性的免疫应答，从而激活肠粘膜效应部位淋巴细胞的抗弓形虫感染作用。

Objective To study the mucosal immune responses of Peyer＇s patches （PP） after intranasal immunization with mucosal vaccine for Toxoplasma gondii, and to observe the duration of the responses. Methods BALB/c mice were randomly divided into two groups： immunized group and control group, 48 mice per group. Mice were intranasally immunized with 20μ1 mucosal complex vaccine （20 μg STAg＋1 μg CT） per mouse twice at an internal of two weeks, while control mice were given PBS solution instead. Six mice of each group were killed respectively on week 1, 2, 3, 4, 6, 8,10 and 12 after the last immunization, respectively. PP was separated and lymphocytes were isolated and counted. Percentage of CD4^＋ and CD8^＋ T cells was determined by immunocytochemistry. Results The number of PP in two groups had no significant changes. Lymphocytes in PP significantly increased after immunity. Lymphocytes in PP of immunized mice were higher than that of control on week 1, 3 （P〈0.05）, 2 （P〈0. 0001）, getting its maximum number on week 2. The CD4^＋ T cell subset was increasing on week 1, 2, 3, 4, 6, 8 （P〈0.01）, while CD8^＋ T cells increased on week 1, 2, 3, 4 （P〈0.01）. And the ratio of CD4^＋ and CD8^＋ Thad no changes. Conclusion Intranasal immunization with mucosal vaccine for T. gondii can effectively induce immune responses of PP to protect mice against T. gondii.