中性粒细胞胞外诱捕网生成和降解方法的建立和评价

Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation

目的中性粒细胞胞外诱捕网(NET)是近年来新发现的与自身免疫性疾病和血栓形成有关的结构,本研究拟建立一种评价NET体外生成和降解的方法。方法通过Ficoll-Histopaque密度梯度离心法从健康人外周血分离中性粒细胞,利用佛波酯(PMA)诱导其产生NET,免疫荧光细胞化学染色和琼脂糖凝胶电泳验证NET的形成,PicoGreen dsDNA染色结合荧光强度进行定量分析,研究DNA酶(DNaseⅠ)和健康人血清对NET的降解。结果 Ficoll密度梯度离心法分离的中性粒细胞纯度达到95%。激光共聚焦显微成像显示NET网状结构主要由DNA和组蛋白组成,琼脂糖凝胶电泳显示NET的DNA条带大小超过10 000 bp。DNase和健康人血清均可降解体外诱导的NET,但程度不同。结论本实验成功建立了人NET体外诱导生成和降解的方法。

Objective To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps（ NETs）,which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis.Methods Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate（ PMA） and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase Ⅰ and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. Results HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones,with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover,both DNase and healthy human plasma could induce the degradation of NETs,in varying degrees. Conclusion This work established an efficient method for in vitro generation and degradation of human NETs.