可溶性虫卵抗原和成虫抗原刺激日本血吸虫感染小鼠肠系膜淋巴结Th17细胞的免疫应答

摘要/Abstract

摘要：

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（100 μg/ml）+ 抗-CD28 mAb（1 μg/ml）（B组）,SWA（100 μg/ml）+ 抗-CD28 mAb（1 μg/ml）（C组）,抗-CD3 mAb（1 μg/ml）+ 抗-CD28 mAb（1 μg/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（A₄₅₀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。

关键词: 日本血吸虫, 肠系膜淋巴结, 可溶性虫卵抗原, 可溶性成虫抗原, Th17细胞

Abstract:

Objective To observe the immune response of mouse Th17 cells from mesenteric lymph node (MLN) after treatment with soluble egg antigens (SEA) and soluble adult antigens (SWA) of Schistosoma japonicum. Methods Two New Zealand white rabbits were infected with S. japonicum cercariae through abdominal skin attachment for 20 min. After 45 days, SWA and SEA were collected. Twenty C57BL/6 mice were randomly divided into the infection group and the healthy group (n = 10 in each group). The mice in the infection group were infected with 40 ± 5 worms of S. japonicum through the abdominal skin. At 5 to 6 weeks, the serum levels of SEA- and SWA-specific IgG were determined by ELISA. The MLN lymphocytes were isolated and divided into four groups. Lymphocytes in group A received no stimulation, those in group B were treated with 100 μg/ml SEA + 1 μg/ml anti-CD28 mAb, those in group C were treated with 100 μg/ml SWA + 1 μg/ml anti-CD28 mAb, and in group D they were treated with 1 μg/ml anti-CD3 mAb + 1 μg/ml anti-CD28 mAb. The levels of IL-17 and IFN-γ in the supernatant were detected by ELISA after 72 h treatment, and Th17 cells were detected by flow cytometry after 9 h treatment. Results ELISA results showed that the A₄₅₀ values for serum SEA- and SWA-specific IgG in the infection group (2.66 ± 0.20, 1.68 ± 0.66) were both significantly higher than those of the healthy mice (0.19 ± 0.05, 0.25 ± 0.12, respectively) (P < 0.01). Flow cytometry showed that the proportions of CD4⁺IL-17⁺ cells and CD4⁺IFN-γ⁺ cells in group B lymphocytes from infected mice (0.43% and 0.56%, respectively) were significantly higher than the healthy group (0.05% and 0.20%) (P < 0.05). Similar results were found for group C (0.39% and 0.76% vs. 0.04% and 0.19%) (P < 0.05). The levels of IFN-γ and IL-17 were (49.13 ± 14.71) and (41.73 ± 2.42) pg/ml, respectively in group B lymphocytes from infected mice, which were significantly higher than the (3.27 ± 0.33) and (9.22 ± 0.58) pg/ml in healthy mice (P < 0.01). Similar results were found in group C [(46.92 ± 16.73) and (36.14 ± 4.82) pg/ml vs. (3.38 ± 0.34) and (8.78 ± 0.93) pg/ml] (P < 0.01). The IL-17 level in group B was (41.72 ± 2.42 ) pg/ml, significantly higher than the (36.14 ± 4.82) pg/ml in group C. Conclusion Both SEA and SWA can induce secretion of IL-17 and IFN-γ from MLN lymphocytes of C57BL/6 mice infected with S. japonicum, and the secreted IL-17 is significantly higher for SEA than SWA. SEA and SWA can also induce the production of CD4⁺IL-17⁺ cells and CD4⁺IFN-γ⁺ cells in the mesenteric lymph nodes of C57BL/6 mice infected with S. japonicum.

Key words: Schistosoma japonicum, Mesenteric lymph node, Soluble egg antigen, Soluble adult antigen, Th17

中图分类号:

R383.24

罗雪平, 袁南贵, 黄俊. 可溶性虫卵抗原和成虫抗原刺激日本血吸虫感染小鼠肠系膜淋巴结Th17细胞的免疫应答[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 105-109.

Xue-ping LUO, Nan-gui YUAN, Jun HUANG. Immune response of mouse Th17 cells from mesenteric lymph node after treatment with soluble egg antigens and adult worm antigens of Schistosoma japonicum[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 105-109.

图/表 3

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