作者简介：危芙蓉（1985-）,女,博士研究生,助理研究员,从事寄生虫病研究。E-mail：
收稿日期: 2018-10-26
网络出版日期: 2019-03-18
基金资助
国家自然科学基金（No. 81772225）;上海市第四轮公共卫生三年行动计划（No. 15GWZK0101)
Leishmania infantum infection stimulates neutrophils to generate extracellular traps in mouse
Received date: 2018-10-26
Online published: 2019-03-18
Supported by
Supported by the National Natural Science Foundation of China(No. 81772225) and the Fourth Round of Three?Year Public Health Action Plan of Shanghai, China(No. 15GWZK0101)
目的 通过定性和定量分析婴儿利什曼原虫感染小鼠体内中性粒细胞被佛波酯刺激诱导产生胞外诱捕网的能力变化。方法 将60只BALB/c小鼠随机均分为感染组和健康对照组,每组30只。感染组每鼠腹腔接种0.5 ml婴儿利什曼原虫前鞭毛体（密度为2 × 108/ml）悬液,12周后乙醚麻醉后收集抗凝血。取感染组和对照组小鼠的抗凝血400 μl加入到24孔板中,用100 nmol/L佛波酯刺激3 h后,分别用CD11b和Ly6G标记中性粒细胞,用H3Cit和MPO标记产生胞外诱捕网的中性粒细胞,流式细胞术检测H3Cit+MPO+中性粒细胞群比例。用中性粒细胞分离试剂盒纯化感染组和对照组小鼠外周血中性粒细胞,100 nmol/L佛波酯刺激中性粒细胞3 h,分别用蓝色荧光标记DNA,红色荧光标记瓜氨酸化组蛋白H3,绿色荧光标记髓过氧化物酶,激光共聚焦显微成像观察、拍照。采用SPSS 19.0统计学软件对数据进行分析,两组间均数比较采用独立样本t检验。结果 采用小鼠外周血中性粒细胞分离试剂盒分离纯化的中性粒细胞纯度达到87%以上。感染组和对照组小鼠的中性粒细胞分别被佛波酯刺激后,H3Cit+MPO+中性粒细胞群占总中性粒细胞的比例分别为（1.880 ± 0.251）%、（0.623 ± 0.087）%,两组差异有统计学意义（P < 0.01）。激光共聚焦显微成象分析发现,感染组的中性粒细胞体积增大,且产生环状和丝状DNA骨架结构的细胞比例增多。感染组标记H3Cit和MPO颗粒物的荧光数量较对照组增多,DNA骨架结构上附着的H3Cit和MPO颗粒物数量增多。结论 婴儿利什曼原虫感染可增强小鼠中性粒细胞产生胞外诱捕网的能力。
危芙蓉 , 杨玥涛 , 汪俊云 , 王燕娟 , 潘佳明 , 曹建平 . 婴儿利什曼原虫感染小鼠体内中性粒细胞形成胞外诱捕网能力的研究[J]. 中国寄生虫学与寄生虫病杂志, 2019 , 37(1) : 18 -22 . DOI: 10.12140/j.issn.1000-7423.2019.01.004
Objective To analyze the effect of Leishmania infantum infection on the differentiation of neutrophil extracellular traps (NETs) of mouse neutrophils under induction of MPA and the possible functions associated with the defense mechanism or pathogenesis of leishmaniasis. Methods Each of 30 BABL/c mice was intraperitoneally infected with 1 × 108 L. infantum promastigotes in total volume of 0.5 ml. The peripheral blood were collected from the ether-anesthetized mice infected with L. infantum for 12 weeks or from normal control mice and the blood cells were stimulated with 100 nmol/L PMA for 3 h to generate NETs. The Red blood cells were lysed and the neutrophils were co-labeled with CD11b and Ly6G and NETs-generating neutrophils were labeled with H3Cit (citrullinated histone 3) and MPO (myeloperoxidase). The neutrophils and NET-generating neutrophils (H3Cit+MPO+) were quantitatively analyzed by flow cytometry. For fluorescent staining, the neutrophils were isolated from peripheral blood and the NET-generating neutrophils were induced with 100 nmol/L PMA for 3 h. The DNA, H3Cit and MPO in the neutrophils were stained with specific antibodies labeled with blue, red and green fluorescence, respectively and visualized under laser scanning confocal microscope. Results L. infantum infection significantly stimulated the differentiation of H3Cit+MPO+ NET neutrophils in the blood of mice after being stimulated with PMA (1.880 + 0.251)% compared to that in the normal control mice (0.623 + 0.087)% with statistical significance (P < 0.01). Laser confocal microscopic imaging analysis showed that the L. infantum infection stimulated more neutrophils of mouse to differentiate to NETs characterized by the filamentous DNA and H3Cit+MPO+ particles attached to the DNA skeleton structure, compared to neutrophils from normal mouse.
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