日本血吸虫感染小鼠肠系膜淋巴结Th17细胞的免疫应答

Immune Response of Th17 Cells in Mesenteric Lymph Node of Mice Infected by Schistosoma japonicum

目的观察C57BL／6小鼠感染日本血吸虫后肠系膜淋巴结Th17细胞的免疫应答。方法20只C57BU6小鼠随机分为感染组和对照组．每组10只，感染组小鼠经腹部皮肤感染日本血吸虫尾蚴，每鼠（40＋5）条。感染后5-6周分离小鼠肠系膜淋巴结的淋巴细胞，分别用抗小鼠CD3单克隆抗体（anti-CD3，1μg／m1）和抗小鼠CD28单克隆抗体（anti-CD28，1μg/ml）刺激，培养4h后收集细胞，RT-PCR检测小鼠肠系膜淋巴结淋巴细胞中自细胞介素17（IL-17）和维甲酸相关孤独受体（ROR-γt）mRNA的转录水平；培养72h后，ELISA检测细胞培养上清液IL-17和1干扰素（IFN-1）的含量。同时用佛波酯（PMA，10ng／m1）和离子霉素（1μg／ml）刺激淋巴细胞5h后，胞内细胞因子染色，流式细胞术检测Th17细胞的含量和其他细胞因子的产生。结果ELISA检测结果显示，感染小鼠肠系膜淋巴结培养物上清液中IFN-γ[（214．3±62．6）pg／ml]和IL-17[（176．8±62．1）pg／ml]的含量明显高于健康小鼠[（46．7±13．9）和0pg／ml]（P〈0．05）。RT-PCR检测结果显示，IL-17和ROR-γt mRNA转录水平也明显高于健康小鼠。感染小鼠肠系膜淋巴细胞CD4＋T细胞中，Th17细胞的比例为（0．55±0．03）％，明显高于健康小鼠[（0．16±0．01）％]（只以05）。在CD4＋T细胞中，IL-17＋ IL4＋细胞占0．06％，IL-17＋IFNγt和IL-17＋IL5＋细胞各占0．02％，IL-17＋ IL-9＋细胞占0.01％，未检测到Tl-17＋IL-10＋和IL-17＋Foxp3＋细胞。结论日本血吸虫感染C57BIJ6小鼠的肠系膜淋巴结能诱导Th17细胞产生。Th17细胞能分泌IL-4，及少量的IFN-γ、IL5和IL-9，不分泌IL-10，也不表达Foxp3。

Objective To observe the immune response of Th17 cells in mes＇enteric lymph node （MLN） of C57BL/6 mice infected by Schistosoma japonicum. Methods Twenty C57BL/6 mice were randomly divided into infected group and control group each with ten mice. The mice in infected group were infected each with 40±5 S. japonicum cercariae. Five to six weeks later, MLN lymphocytes were separated and stimulated for 4 h by anti-CD3 （1μg/ml） and anti-CD28 （1μg/ml） before examination of IL-17 and retinoic acid receptor-related orphan receptor γt （ROR-γt） mRNA by reverse transcription PCR. The level of IL-17 and IFN-γ was detected by ELISA after culturing with supernatant for 72 h. MLN lymphoeytes were stimulated for 5 h by 10ng/ml phorbol myristoyl acetate （PMA） and 1 μg/ml ionomycine. The intracellular cytokines were stained and the content of Thl7 and other cytokines was examined by flow cytometry. Results The level of IFN-γ[（214.3±62.6） pg/ml] and IL-17 [（176.8±62.1） pg/ml] in the supernatant of culured MLN cells from the infected mice was significantly higher than that of normal mice [（46.7±13.9） and 0 pg/ml）（P〈0.05）. The expression level of IL-17 and ROR-γt mRNA was also considerably higher than that of normal mice. IL-17＋ IL-4＋, IL-17＋IFN-γt, IL-17＋ IL-5＋ and IL-17＋ IL-9 cells accounted for 0.06%, 0.02%, 0.02%, and 0.01% of the mesenteric lymph node CD4＋ T cells of the infected mice, respectively. However, IL-17＋ IL-10＋ and IL-17＋ Foxp3＋ cells were undetected. Conclusion The MLN of S. japonicum-infected C57BL/6 mice can induce the production of Thl7 cells, and these cells can secrete IL-4, less IFN-γ, IL-5 and IL-9, but not IL-10, and can not express Foxp3 in the infected mice.