[百草枯体外诱导人外周血中性粒细胞胞外诱捕网形成的实验研究]
[Experimental study on formation of neutrophil extracellular traps in human peripheral blood induced by paraquat in vitro].
作者信息
Jin Wei, Lu Jian, Xie Hui, Jiang Yiqun, Meng Xiaoxiao, Zhu Yong, Wang Ruilan
机构信息
Department of Critical Care Medicine, Shanghai General Hospital of Nanjing Medical University, Shanghai 200080, China. Corresponding author: Wang Ruilan, Email:
出版信息
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Mar;29(3):216-220. doi: 10.3760/cma.j.issn.2095-4352.2017.03.005.
OBJECTIVE
To explore whether paraquat (PQ) can induce the formation of neutrophil extracellular traps (NETs) in human peripheral blood.
METHODS
Neutrophils were isolated from healthy human peripheral blood, and the cells were identified by hematoxylin-eosin (HE) strain. The cells were treated with different concentrations of PQ [0 (as control), 200, 400, 600, 800, 1 000 and 1 200 μmol/L], and the cell viability was measured by cell proliferation and CCK-8 cytotoxicity detection kit, and the median lethal concentration of PQ was selected. The cells were treated with the median lethal concentration of PQ (PQ poisoning group), and the untreated cells were served as the control. Immunofluorescence staining was adopted to evaluate NETs formation. PicoGreen dye was used to determine the quantitative content of circulating free DNA. Western Blot was used to determine the expressions of citrullinated histone 3 (H3Cit) and myeloperoxidase (MPO) in the supernatant.
RESULTS
The purity of neutrophils was about 95% by HE staining. The cells were treated with different concentrations of PQ, and the result showed that the viability of cells was (58±2)% with 800 μmol/L PQ for treatment. The immunofluorescence showed that there were few expressions of H3Cit and MPO in neutrophils in the control group, and there was no NETs formation, which was composed of DNA, H3Cit and MPO. Compared with the control group, a large amount of NETs was generated from neutrophils stimulated by 800 μmol/L of PQ. Meanwhile, quantitative result showed that the content of cell free DNA in the supernatant was significantly increased in PQ poisoning group as compared with that of control group (μg/L: 2 235±462 vs. 561±87, P < 0.01). The protein expressions of H3Cit and MPO in the supernatant were also significantly increased as compared with those of control group [H3Cit protein expression (gray value): 0.23±0.03 vs. 0.11±0.01, MPO protein expression (gray value): 0.47±0.05 vs. 0.21±0.04, both P < 0.05].
CONCLUSIONS
800 μmol/L of PQ can induce the formation of NETs in human peripheral blood.
目的
探讨百草枯（PQ）是否能诱导人外周血中性粒细胞胞外诱捕网（NETs）的形成。
方法
从健康人外周血中分离中性粒细胞，并用苏木精-伊红（HE）染色鉴定细胞。将细胞用不同浓度的PQ[0（作为对照）、200、400、600、800、1000和1200μmol/L]处理，通过细胞增殖和CCK-8细胞毒性检测试剂盒检测细胞活力，并选择PQ的半数致死浓度。将细胞用PQ的半数致死浓度处理（PQ中毒组），未处理的细胞作为对照。采用免疫荧光染色评估NETs的形成。用PicoGreen染料测定循环游离DNA的定量含量。用蛋白质免疫印迹法检测上清液中瓜氨酸化组蛋白3（H3Cit）和髓过氧化物酶（MPO）的表达。
结果
HE染色显示中性粒细胞纯度约为95%。用不同浓度的PQ处理细胞，结果显示800μmol/L PQ处理后细胞活力为（58±2）%。免疫荧光显示对照组中性粒细胞中H3Cit和MPO表达较少，无由DNA、H3Cit和MPO组成的NETs形成。与对照组相比，800μmol/L PQ刺激的中性粒细胞产生大量NETs。同时，定量结果显示，PQ中毒组上清液中游离DNA含量较对照组显著增加（μg/L：2235±462比561±87，P<0.01）。上清液中H3Cit和MPO的蛋白表达也较对照组显著增加[H3Cit蛋白表达（灰度值）：0.23±0.03比0.11±0.01，MPO蛋白表达（灰度值）：0.47±0.05比0.21±0.04，均P<0.05]。
结论
800μmol/L的PQ能诱导人外周血中NETs的形成。
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