目的 动态观察细粒棘球绦虫(Eg)重组双歧杆菌(Bb)-Eg95-EgA31疫苗免疫小鼠后脾细胞因子的变化.方法 将5×108克隆形成单位(CFU)和5×105 CFU疫苗分别采用口服灌胃和鼻腔黏膜接种免疫Balb/c小鼠,在免疫后0、2、4、6、8、10、12、14、16、18、20周各剖杀4只小鼠,取脾,分离脾细胞,用Eg粗抗原(EgAg)、刀豆蛋白A(ConA)或脂多糖(LPS)刺激培养,同时设有双歧杆菌液体培养基(MRS)对照.收集脾细胞培养上清液,ELISA法检测γ干扰素(IFN-γ)、白细胞介素(IL)-12、肿瘤坏死因子α(TNF-α)和IL-10水平.结果 口服灌胃组的IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2～8、2～8、4、6～10周升高,分别在免疫后4、2、4、6周达最高水平,其EgAg刺激组IFN-γ、IL-12、TNF-α和IL-10水平分别为(700.0±115.5)、(45.0±5.8)、(350.0±57.7)、(112.5±14.4)ng/L,与0周[(35.0±5.8)、(12.5±2.9)、(190.0±11.6)、(25.0±5.8)ng/L]及MRS对照组[(37.5±5.0)、(13.8±2.5)、(195.0±5.8)、(27.5±2.9)ng/L]比较,差异有统计学意义(P＜0.05或＜0.01);鼻腔接种组的IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2～8、2～8、2～6、6～16周升高,分别在免疫后2、2、4、8周达最高水平,其EgAg刺激组IFN-γ、IL-12、TNF-α和IL-10水平分别为(700.0±115.5)、(55.0±5.8)、(275.0±28.9)、(140.0±11.6)ng/L,与0周[(35.0±5.8)、(12.5±2.9)、(190.0±11.6)、(25.0±5.8)ng/L]及MRS对照组[(37.5±5.0)、(13.8±2.5)、(195.0±5.8)、(27.5±2.9)ng/L]比较,差异有统计学意义(P＜0.05或＜0.01).EgAg、ConA、LPS刺激组的细胞因子水平高于相应的脾细胞悬液组(P＜0.05或＜0.01),ConA或LPS刺激组的细胞因子水平高于相应的EgAg刺激组(P＜0.05或＜0.01).结论 细粒棘球绦虫重组Bb-Eg95-EgA31疫苗在免疫早期(2～6周)可诱导小鼠脾细胞产生Th1和Th2混合型免疫应答.%Objective To dynamically observe the changes of cytokines of splenocytes in mice immunized with recombinant bifidobacteria bifidum (Bb)- Eg95-EgA31 vaccine of Echinococcus grauulosus (Eg). Methods Balb/c mice were vaccinated by 5× 108 colony forming unit(CFU) orally and 5 × 105 CFU intranasally, respectively.Mice were killed on week 0,2,4,6,8,10, 12,14,16, 18 and 20 after immunization, respectively, and spleens were separated for cell culture with the stimulation of EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS). The splenocyte supernatants were collected to determine the levels of interferonγ(IFN-γ), interleukin(IL)-12, tumor necrosis factor α(TNF-o) and IL-l0 using enzyme linked immunosorbent assay(ELISA) with MRS as control. Results In the oral immunization group, the levels of IFN-γ, IL-12, TNF-α and IL-10 showed a significant increase from week 2 to week 8, week 2 to week 8, week 4 and week 6 to week 10 after vaccination, respectively, and reached the highest level on week 4, week 2, week 4 and week 6 after vaccination, respectively;in EgAg stimulation group, the levels of IFN-γ, IL-12, TNF-α and IL-10 were (700.0 ± 115.5), (45.0 ± 5.8), (350.0 ± 57.7), (112.5 ± 14.4)ng/L, respectively, compared with week 0[(35.0 ± 5.8), (12.5 ± 2.9), (190.0 ± 11.6), (25.0 ± 5.8)ng/L, P ＜0.05 or ＜ 0.01] and MRS control group[(37.5 ± 5.0),(13.8 ± 2.5), (195.0 ± 5.8), (27.5 ± 2.9)ng/L, P＜ 0.05or ＜ 0.01]. In the intranasal immunization group, the levels of IFN-γ, IL-12, TNF-α and IL-10 showed an obvious increase from week 2 to week 8, week 2 to week 8, week 2 to week 6 and week 6 to week 16 after vaccination,respectively, and reached the highest level on week 2, week 2, week 4 and week 8 after vaccination, respectively;in EgAg stimulation group, the levels of IFN-γ, IL-12, TNF-α and IL-10 were (700.0 ± 115.5), (55.0 ± 5.8),(275.0 ± 28.9), (140.0 ± 11.6)ng/L, compared with week 0[(35.0 ± 5.8), (12.5 ± 2.9), (190.0 ± 11.6), (25.0 ±5.8)ng/L, P ＜ 0.05 or ＜ 0.01] and MRS control group[(37.5 ± 5.0), (13.8 ± 2.5), (195.0 ± 5.8), (27.5 ± 2.9)ng/L, P ＜ 0.05 or ＜ 0.01]. The cytokine levels in the groups with EgAg, ConA or LPS stimulus were significantly higher than those in the corresponding splenocytes suspension groups(P ＜ 0.05 or ＜ 0.01) , and the cytokine levels in the groups with ConA or LPS stimulus were obviously higher than those in the corresponding groups with EgAg stimulation(P ＜ 0.05 or ＜ 0.01). Conclusion The mixed Th1 and Th2 type response can be induced in mice immunized with the recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus in the early stage of immunization(2 to 6weeks).
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