体外诱导中性粒细胞胞外诱捕网形成条件的优选及验证  

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

作　　者：戚明珠 李卓航 黄荷兰 祝盼盼 朱鏐娈[2] 林娜[1] 苏晓慧[1] 孔祥英[1] QI Ming-zhu;LI Zhuo-hang;HUANG He-lan;ZHU Pan-pan;ZHU Liu-luan;LIN Na;SU Xiao-hui;KONG Xiang-ying(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Beijing Ditan Hospital Affiliated to Capital Medical University,Beijing 100015,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]首都医科大学附属北京地坛医院,北京100015

出　　处：《中国中药杂志》2024年第9期2336-2344,共9页China Journal of Chinese Materia Medica

基　　金：中国中医科学院科技创新工程创新团队项目(CI2021B004)。

摘　　要：旨在优选体外诱导中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)形成的条件,以建立相对稳定的实验技术平台。通过比较不同条件,包括大鼠和小鼠等常用的实验动物,利用percoll密度梯度离心法分离骨髓中性粒细胞、外周血中性粒细胞等多种细胞来源,采用脂多糖(lipopolysaccharide,LPS)和佛波醇12-肉豆蔻酸酯13-乙酸酯(phorbol 12-myristate 13-acetate,PMA)不同诱导剂体外诱导,通过MPO/CitH3/DAPI免疫荧光标记法以及细胞培养上清游离DNA(cell free DNA,cfDNA)含量检测综合评价,筛选体外诱导NETs形成的优选条件;在上述平台选择基础上,进一步通过给予中药有效单体川芎嗪(tetramethylpyrazine,TMP)进行验证,综合评估优选的体外NETs诱导条件的稳定性。结果显示,包被多聚赖氨酸可一定程度促进小鼠骨髓中性粒细胞NETs形成。LPS和PMA均可显著促进小鼠骨髓中性粒细胞MPO、CitH3蛋白表达,并提高大鼠外周血中性粒细胞培养上清中cfDNA含量;且与LPS相比,PMA诱导cfDNA水平升高更显著(P<0.05)。与PMA共孵育后小鼠骨髓中性粒细胞、大鼠骨髓中性粒细胞和大鼠外周血中性粒细胞中MPO、CitH3蛋白表达均显著升高,以大鼠外周血中性粒细胞升高最明显。TMP可以显著下调PMA诱导的大鼠外周血中性粒细胞中MPO、CitH3和NE蛋白的表达。综上,PMA诱导大鼠外周血中性粒细胞是体外诱导NETs形成的较优条件。该研究为基于NETs的体外研究提供优选平台,并为以调控NETs为靶标的中药作用研究提供前期基础。This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs)in vitro,so as to es-tablish a relatively stable experimental research platform.Different conditions were compared,including commonly used laboratory ani-mals(rats and mice)and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll den-sity gradient centrifugation).Different inducers like lipopolysaccharide(LPS)and phorbol 12-myristate 13-acetate(PMA)were used for induction in vitro.Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA)content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro.Furthermore,the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetrameth-ylpyrazine(TMP),an active component in Chinese herbal medicines.The results showed that coated poly-D-lysine(PDL)induced the formation of NETs in bone marrow neutrophils of mice to a certain extent.Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils.The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05).The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils,rat bone marrow neutrophils,and rat peripheral blood neutrophils were significantly increased after PMA induction,especially in rat peripheral blood neutrophils.TMP significantly down-regulated the protein levels of MPO,CitH3,and neutrophil elastase(NE)in rat peripheral blood neutrophils induced by PMA.In conclusion,treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro.This study provides an optimal plaform for in vitro studies based on NETs an

关 键 词：中性粒细胞胞外诱捕网 免疫荧光 骨髓 外周血 川芎嗪 

分 类 号：R228[医药卫生—中医基础理论]