活性氧在Anti-β2GPⅠ/β2GPⅠ诱导中性粒细胞外诱捕网产生中的作用机制研究  

Study on the mechanism of reactive oxygen species in Anti-β2GPⅠ/β2GPⅠ-induced the formation of neutrophil extracellular traps

作　　者：徐佳丽[1] 张丽梅[1] 刘彦虹[2] Xu Jiali;Zhang Limei;Liu Yanhong(Laboratory of Endocrinology and Metabolism Department,the Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China;Department of Clinical Laboratory,the Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China)

机构地区：[1]哈尔滨医科大学附属第二医院内分泌实验室,150086 [2]哈尔滨医科大学附属第二医院检验科,150086

出　　处：《国际免疫学杂志》2019年第6期553-557,共5页International Journal of Immunology

基　　金：国家自然科学基金(81541031)。

摘　　要：目的探讨活性氧(reactive oxygen species,ROS)在抗β2糖蛋白Ⅰ(β2 GlycoproteinⅠ,β2GPⅠ)抗体/β2GPⅠ通过相关信号转导通路诱导中性粒细胞外诱捕网(neutrophil extracellular traps,NETs)形成中的重要作用.方法提取健康志愿者中性粒细胞,PBS刺激作为PBS组、anti-β2GPⅠ/β2GPⅠ(10/100μg/mL)刺激作为anti-β2GPⅠ/β2GPⅠ组、anti-β2GPⅠ/β2GPⅠ+还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶抑制剂(DPI 20μmol/L)刺激作为anti-β2GPⅠ/β2GPⅠ+DPI组、anti-β2GPⅠ/β2GPⅠ+p38丝裂原活化蛋白激酶(mitogen activatedprotein kinase,MAPK)抑制剂(SB20358010μmol/L)刺激作为anti-β2GPⅠ/β2GPⅠ+SB203580组、anti-β2GPⅠ/β2GPⅠ+细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)抑制剂(U012610μmol/L)刺激作为anti-β2GPⅠ/β2GPⅠ+U0126组、佛波酯(phorbol-12-myristate-13-acetate,PMA)(50 nmol/L)刺激作为PMA组.流式细胞仪检测中性粒细胞ROS的释放;Western blot检测中性粒细胞p38MAPK、ERK活化.结果与anti-β2GPⅠ/β2GPⅠ组相比,anti-β2GPⅠ/β2GPⅠ+DPI组、anti-β2 GPⅠ/β2GPⅠ+SB203580组、anti-β2GPⅠ/β2GPⅠ+U0126组活性氧释放百分率减少,且anti-β2 GPⅠ/β2GPⅠ+DPI组抑制作用更明显[anti-β2GPⅠ/β2GPⅠ组比anti-β2GPⅠ/β2GPⅠ+DPI组:(35.21%±1.22%)比(1.62%±0.08%),P<0.05;anti-β2GPⅠ/β2GPⅠ组比Anti-β2GPⅠ/β2GPⅠ+SB203580组:(35.21%±1.22%)比(10.21%±0.98%),P<0.05;anti-β2GPⅠ/β2GPⅠ组比anti-β2GPⅠ/β2GPⅠ+U0126组:(35.21%±1.22%)比(8.36%±0.62%),P<0.05)].Anti-β2GPⅠ/β2GPⅠ+SB203580组、anti-β2GPⅠ/β2GPⅠ+U0126组分别使p38 MAPK、ERK磷酸化水平下降(anti-β2GPⅠ/β2GPⅠ+SB203580组比anti-β2GPⅠ/β2GPⅠ组:[(0.601±0.031)比(1.212±0.132),P<0.05;anti-β2GPⅠ/β2GPⅠ+U0126组比anti-β2GPⅠ/β2GPⅠ组:(0.107±0.001)比(1.612±0.096),P<0.05)],而anti-β2GPⅠ/β2GPⅠ+DPI组p38 MAPK、ERK磷酸化水平均无明显下降[anti-β2GPⅠ/β2GPⅠ+DPI�Objective The purpose of this study is to investigate the important role of reactive oxygen species(ROS)in anti-β2 GlycoproteinⅠ(β2GPⅠ)/β2GPⅠ-induced the formation of neutrophil extracellular traps(NETs)by related signal transduction pathway.Methods Extraction of healthy volunteer neutrophils.According to the PBS stimulation as PBS group,anti-β2GPⅠ/β2GPⅠ(10/100 g/mL)stimulus as anti-β2GPⅠ/β2GPⅠgroup,anti-β2GPⅠ/β2GPⅠ+nicotinamide adenine dinucleotide phosphate(NADPH)oxidase inhibitor(DPI 20μmol/L)stimulus as anti-β2GPⅠ/β2GPⅠ+DPI group,anti-β2GPⅠ/β2GPⅠ+p38mitogen activated protein kinase(MAPK)inhibitor(SB20358010μmol/L)stimulus as anti-β2GPⅠ/β2GPⅠ+SB203580group,anti-β2GPⅠ/β2GPⅠ+extracellular regulated protein kinases(ERK)inhibitor(U012610μmol/L)stimulus as anti-β2GPⅠ/β2GPⅠ+U0126 group,andphorbol-12-myristate-13-acetate(PMA)(50 nmol/L)stimulation as PMA group.Flow cytometry detected the release of reactive oxygen species in neutrophils;Western blot detected the activation of p38MAPK and ERK in neutrophils.Results Compared to the anti-β2GPⅠ/β2GPⅠstimulated group,anti-β2GPⅠ/β2GPⅠ+DPI group、anti-β2GPⅠ/β2GPⅠ+SB203580 group、anti-β2GPⅠ/β2GPⅠ+U0126 group decreased the release of ROS and anti-β2GPⅠ/β2GPⅠ+DPI group had more obvious inhibition[anti-β2GPⅠ/β2GPⅠvs anti-β2GPⅠ/β2GPⅠ+DPI:(35.21%±1.22%)vs(1.62%±0.08%),P<0.05;anti-β2GPⅠ/β2GPⅠvs Anti-β2GPⅠ/β2GPⅠ+SB203580:(35.21%±1.22%)vs(10.21%±0.98%),P<0.05;anti-β2GPⅠ/β2GPⅠvs anti-β2GPⅠ/β2GPⅠ+U0126:(35.21%±1.22%)vs(8.36%±0.62%),P<0.05].Anti-β2GPⅠ/β2GPⅠ+SB203580 group、anti-β2GPⅠ/β2GPⅠ+U0126 group decreased the phosphorylation level of p38MAPK,ERK respectively(anti-β2GPⅠ/β2GPⅠ+SB203580 vs anti-β2GPⅠ/β2GPⅠ:(0.601±0.031)vs(1.212±0.132),P<0.05;anti-β2GPⅠ/β2GPⅠ+U0126vs anti-β2GPⅠ/β2GPⅠ:(0.107±0.001)vs(1.612±0.096),P<0.05),p38MAPK and ERK phosphorylation levels were not significantly decreased in anti-β2GPⅠ/β2G

关 键 词：中性粒细胞胞外诱捕网 Anti-β2GPⅠ/β2GPⅠ复合物 活性氧 

分 类 号：R73[医药卫生—肿瘤]