摘要
【目的】采用定性与定量方法探讨甲型流感病毒(influenza A virus,IAV)H1N1亚型诱导中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)的形成水平。【方法】分离、纯化并鉴定小鼠骨髓中性粒细胞,使用脂多糖(lipopolysaccharide,LPS)和佛波酯(phorbol 12-myristate 13-acetate,PMA)作为诱导剂在体外诱导NETs形成。同时设置3个滴度的甲型流感病毒组,分别为100个50%组织培养感染剂量(one hundred 50%tissue culture infective doses,100 TCID50)、50 TCID50、25 TCID50和正常对照组。采用RT-qPCR法检测甲型流感病毒组细胞内核衣壳蛋白信使RNA(nucleoprotein messenger RNA,NP mRNA)的表达水平;通过SYTOX Green核酸染料(SYTOX green nucleic acid stain,SYTOX Green)定量法检测各因素干预中性粒细胞后各组上清液中游离DNA(cellfree DNA,cfDNA)的含量;采用Hoechst 33342染色法,在荧光显微镜下观察各组细胞形成的NETs结构;运用免疫荧光法检测NETs的特征性指标瓜氨酸化组蛋白(citrullinated histone H3,CitH3)、肽基精氨酸脱亚氨酶4(peptidylarginine deiminase 4,PAD4)、髓过氧化物酶(myeloperoxidase,MPO)、中性粒细胞弹性蛋白酶(neutrophil elastase,NE)蛋白的表达水平,以及各组细胞中PAD4与CitH3、MPO与NE的核共定位和荧光表达强度;使用荧光探针法检测细胞活性氧(reactive oxygen species,ROS)水平;采用Western blotting法检测细胞内CitH3蛋白的形成水平。【结果】从小鼠骨髓中分离的中性粒细胞活性可达98%,纯度可达87%以上。与正常对照组相比,甲型流感病毒组(IAV)的NP mRNA表达水平显著升高;PMA组、LPS组以及甲型流感病毒组(IAV)的cfDNA水平显著升高,NETs网状结构明显增多。免疫荧光结果显示,MPO、NE、PAD4、CitH3蛋白的相对表达量均有不同程度升高,共定位情况相对增多。其中,MPO蛋白表达的高峰期早于NE蛋白,CitH3蛋白表达趋势与PAD4蛋白表达趋势相似。此外,ROS水平升高,CitH3蛋白形成水平显著升高。【结论】甲型流感病毒(H1N1)刺激中性粒细胞后可诱导NETs形成,这可能与细胞内ROS及PAD4水平增加有关。
[Objective]The formation of neutrophil extracellular traps(NETs)induced by influenza A virus(IAV)subtype H1N1 was investigated both qualitatively and quantitatively.[Methods]Mouse bone marrow neutrophils were isolated,purified,and characterized.NETs were induced in vitro using lipopolysaccharide(LPS)and phorbol 12-myristate 13-acetate(PMA).Additionally,IAV groups with three different titers:one hundred 50%tissue culture infective doses(100 TCID50),50 TCID50,and 25 TCID50 as well as the normal control group were established,and the intracellular nucleoprotein(NP)mRNA expression levels of the IAV groups were detected using reverse transcription quantitative polymerase chain reaction(RT-qPCR).The effect of each factor on neutrophils was assessed by measuring the concentration of circulating cell-free DNA(cfDNA)in the supernatant of each group using the quantitative SYTOX Green staining method.The NETs structure in each group of cells was observed under a fluorescence microscope after Hoechst 33342 staining.An immunofluorescence assay was performed to detect the expression levels of NET characteristic markers citrullinated histone H3(CitH3),peptidylarginine deiminase 4(PAD4),myeloperoxidase(MPO),and neutrophil elastase(NE)proteins,as well as the nuclear co-localization and fluorescence intensity of PAD4 with CitH3,and MPO with NE in each group.The levels of reactive oxygen species(ROS)were determined by using a fluorescent probe assay,and the levels of intracellular CitH3 protein formation were determined by using Western blotting.[Results]The activity of neutrophils isolated from mouse bone marrow reached 98%,with purities of≥87%.The expression levels of NP mRNA in the IAV groups were significantly higher than those in the control group.Compared with the control group,the cfDNA levels of the PMA,LPS,and IAV groups were significantly increased,with significant increases in the web-like structures of NETs.The immunofluorescence assay showed that the relative expression levels of MPO,NE,PAD4,and CitH3 proteins were elevated to varying degrees,with the co-localization of PAD4/CitH3 or MPO/NE increased after IAV infection.Moreover,the peak of MPO protein expression was observed before that of NE protein,whereas CitH3 expression paralleled that of PAD4 protein.Additionally,the ROS level was elevated,and the level of CitH3 protein formation was also significantly increased.[Conclusion]Stimulation of neutrophils by IAV(H1N1)induces NET formation,which may be related to the increased intracellular ROS and PAD4 levels.
作者
刘会会
陈纯静
王小奇
梁馨丹
王智槟
张园园
卢芳国
LIU Huihui;CHEN Chunjing;WANG Xiaoqi;LIANG Xindan;WANG Zhibin;ZHANG Yuanyuan;LU Fangguo(Medical School,Hunan University of Chinese Medicine,Changsha,Hunan,China;School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha,Hunan,China)
出处
《微生物学报》
北大核心
2025年第12期5406-5423,共18页
Acta Microbiologica Sinica
基金
国家自然科学基金(82374266,82405375)。
