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

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(P0.05)。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。

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

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 nMPMA 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 nMfor 4 h. However, NETswas formed by neutrophils treated with PMA at 90 nMfor 3 h (P<0.05). NETs release reached the peak when it was treated with 90nMPMA for 4 hours, which was significantly higher than 30 nMfor 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.Conclusion:Neutrophils treated by 90 nMPMA for 4 hr released a large amount of NETs which consistsed of ROS and Elastase.