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目的研究可溶性虫卵抗原(SEA)和可溶性成虫抗原(SWA)刺激日本血吸虫(Schistosoma japonicum)感染小鼠肠系膜淋巴结(MLN)Th17细胞的免疫应答。方法新西兰大白兔2只,经腹部皮肤感染日本血吸虫尾蚴(约1 000条/兔),45 d后收集成虫及虫卵,制备SWA和SEA。20只C57BL/6小鼠按随机数字表法随机分为感染组和健康组(10只/组),感染组小鼠经腹部皮肤感染日本血吸虫尾蚴,(40±5)条/鼠,感染后5~6周ELISA检测血清中SEA和SWA特异性IgG抗体。分离两组小鼠MLN中的淋巴细胞,均给予4种不同条件刺激:无刺激组(A组),SEA(100μg/ml)+抗-CD28 mAb(1μg/ml)(B组),SWA(100μg/ml)+抗-CD28 mAb(1μg/ml)(C组),抗-CD3 mAb(1μg/ml)+抗-CD28 mAb(1μg/ml)(D组)。培养9 h后,流式细胞仪检测Th17细胞;培养72 h后,ELISA检测细胞培养上清中的白细胞介素(IL-17)和γ干扰素(IFN-γ)。结果 ELISA检测结果显示,感染组小鼠血清SEA、SWA特异性IgG抗体的吸光度(A_(450)值)分别为2.66±0.20和1.68±0.66,高于健康组的0.19±0.05和0.25±0.12(P<0.01)。流式细胞术检测结果显示,B组刺激后,感染组淋巴细胞中CD4~+IL-17~+和CD4~+IFN-γ~+细胞分别占0.43%和0.56%,高于健康组的0.05%和0.20%(P<0.05);C组刺激后则分别占0.39%和0.76%,高于健康组的0.04%和0.19%(P<0.05)。两组小鼠的淋巴细胞体外经SEA(B组)刺激后,感染组淋巴细胞产生的IFN-γ和IL-17分别为(49.13±14.71)和(41.73±2.42)pg/ml,高于健康组的(3.27±0.33)和(9.22±0.58)pg/ml(P<0.01);经SWA(C组)刺激后则分别为(46.92±16.73)和(36.14±4.82)pg/ml,高于健康组的(3.38±0.34)和(8.78±0.93)pg/ml(P<0.01)。B组的IL-17含量高于C组(P<0.05)。结论SEA和SWA能体外诱导日本血吸虫感染C57BL/6小鼠肠系膜淋巴结的淋巴细胞分泌IL-17和IFN-γ,SEA刺激淋巴细胞产生IL-17的含量明显高于SWA;SEA和SWA均能诱导淋巴细胞分化为CD4~+IL-17~+细胞和CD4~+IFN-γ~+细胞。
Objective To investigate the immune response of soluble myeloid antigen (SEA) and soluble adult worm antigen (SWA) to Th17 cells in mice infected with Schistosoma japonicum. Methods Two New Zealand white rabbits were infected with cercariae of Schistosoma japonicum (about 1,000 per rabbit) via abdominal skin. After 45 d, adult and insect eggs were collected to prepare SWA and SEA. 20 C57BL / 6 mice were randomly divided into infection group and healthy group (10 / group) according to random number table. Infection group mice were infected with cercariae of Schistosoma japonicum (40 ± 5) / mouse via abdominal skin, 5 to 6 weeks ELISA detection of serum SEA and SWA-specific IgG antibodies. The lymphocytes in MLN of mice were separated and stimulated by four different conditions: group A, SEA 100μg / ml, anti-CD28 mAb 1μg / ml (group B), SWA (group A) 100 μg / ml) + anti-CD28 mAb (group C), anti-CD3 mAb (1 μg / ml) + anti-CD28 mAb (1 μg / ml) After cultured for 9 h, Th17 cells were detected by flow cytometry. After cultured for 72 h, the levels of interleukin (IL-17) and interferon gamma (IFN-γ) in the cell culture supernatants were detected by ELISA. Results The results of ELISA showed that the absorbance (A_ (450)) of SEA and SWA specific IgG in sera from infected mice were 2.66 ± 0.20 and 1.68 ± 0.66, respectively, which were higher than those of healthy group (0.19 ± 0.05 and 0.25 ± 0.12 P <0.01). The result of flow cytometry showed that the percentage of CD4 ~ + IL-17 ~ + and CD4 ~ + IFN-γ ~ + cells in the infected group were 0.43% and 0.56% respectively, which were significantly higher than those in healthy group % And 0.20%, respectively (P <0.05). After stimulation, C group was 0.39% and 0.76% respectively, which was higher than 0.04% and 0.19% of healthy group (P <0.05). After stimulated by SEA (group B), the levels of IFN-γ and IL-17 in the lymphocytes of the two groups were (49.13 ± 14.71) and (41.73 ± 2.42) pg / ml, respectively, which were higher than those of healthy (3.27 ± 0.33) and (9.22 ± 0.58) pg / ml, respectively (P <0.01). After stimulated by SWA (C), they were (46.92 ± 16.73) and (36.14 ± 4.82) pg / (3.38 ± 0.34) and (8.78 ± 0.93) pg / ml for healthy group (P <0.01). The level of IL-17 in group B was higher than that in group C (P <0.05). Conclusions SEA and SWA can induce the secretion of IL-17 and IFN-γ from lymphocytes of C57BL / 6 mice infected with Schistosoma japonicum in vitro, and the production of IL-17 by SEA is significantly higher than that of SWA. SEA and SWA can induce both SEA and SWA Lymphocytes differentiate into CD4 ~ + IL-17 ~ + cells and CD4 ~ + IFN-γ ~ + cells.