| 祝元锋 陈晓丽 鲁永玲 范士郡 刘鑫.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[J].,2016,16(9):1627-1630 |
| 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 NeutrophilExtracellular Traps(NETS) and the Components Research of NETS |
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| DOI： |
| 中文关键词: 佛波酯 中性粒细胞胞外诱捕网 方法 组成结构 |
| 英文关键词: PMA Neutrophil Extracellular Traps Methods Components |
| 基金项目: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（cstc2014jcyjA10068） |
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| 中文摘要: |
| 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 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. |
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