PMA诱导人中性粒细胞胞外诱捕网(NETS)形成的方法及其结构的研究  

Establishment of a Method for PMA induced Formation of Neutrophil Extracellular Traps (NETS) and the Components Research of NETS

作　　者：祝元锋[1] 陈晓丽[1] 鲁永玲[1] 范士郡 刘鑫[1] 

机构地区：[1]第三军医大学第一附属医院综合实验研究中心,重庆400038

出　　处：《现代生物医学进展》2016年第9期1627-1630,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81202566);重庆市前沿与应用基础研究项目(cstc2014jcyj A10068)

摘　　要：目的:建立佛波酯(PMA)诱导人中性粒细胞NETS形成的方法,并研究NETS的结构组成。方法:提取人中性粒细胞,使用10、30、90 n M的PMA分别刺激细胞2、3、4 h,采用核酸染料sytox green染色后,通过共聚焦显微镜检测和比较各组NETS的形成情况,并通过活性氧(ROS)探针对NETS进行荧光染色,对弹性蛋白酶(Elastase)、髓过氧化物酶(MPO)和组蛋白H3(Histone H3)进行免疫荧光染色。结果:PMA低于30 n M刺激细胞4 h都不会产生NETS,90 n M刺激3 h就会形成NETS,使用90 n M刺激中性粒细胞4 h后,其形成的NETS含量最高,显著高于30 n M刺激4 h及90 n M刺激3h(P<0.05)。免疫荧光染色结果显示NETS结构上含有大量ROS和Elastase,含有少量MPO,几乎不含Histone H3。结论:90 n M PMA刺激中性粒细胞4 h可促进NETS形成,其含有大量ROS和Elastase。Objective： To establish an in vitro method for the formation of neutrophil extracellular traps（NETs） induced by phorbol myristate acetate（PMA） and analyze the structure and components of NETs. Methods： Neutrophils were isolated from peripheral blood of healthy donors and treated with 10, 30 and 90 n M PMA for 2, 3 and 4 h respectively. NETs release and its structure were imaged by staining with sytox green and observed under a laser confocal microscopy. Its components were further evaluated by staining with the ROS probe and immunofluorescence staining of Elastase, MPO, and Histone H3. Results： NETs release could not be induced by phorbol myristate acetate（PMA） under 30 n M for 4 h. However, NETswas formed by neutrophils treated with PMA at 90 n M for 3 h（P〈0.05）.NETs release reached the peak when it was treated with 90 n M PMA for 4 hours, which was significantly higher than 30 n M for 4 h or 90 nm for 3 h（P〈0.05）. Immunofluorescence staining showed elevated levels of ROS and high accumulation of elastase in NETs. However,there was minimun accumulation of MPO and Histone H3 was not detected. Conclusions： Neutrophils treated by 90 n M PMA for 4 hr released a large amount of NETs which consistsed of ROS and Elastase.

关 键 词：佛波酯 中性粒细胞胞外诱捕网 方法 组成结构 

分 类 号：R-33[医药卫生] R331.142