欢迎访问《现代生物医学进展》编辑部！

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 nM 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 nM刺激细胞4 h 都不会产生NETS，90 nM刺激3 h就会形成NETS，使用90 nM刺激中性粒细胞4 h后，其形成的NETS 含量最高，显著高于30 nM刺激4 h及90 nM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 nMPMA 刺激中性粒细胞4 h可促进NETS形成，其含有大量ROS 和Elastase。

英文摘要:

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.

View Full Text View/Add Comment Download reader