IL-1β诱导支气管上皮细胞高迁移率族蛋白1主动释放

Inducing effect of IL-1β about the release of HMGB1 in human bronchial epithelial cells

目的研究IL-1β对健康人支气管上皮细胞(HBE)高迁移率族蛋白1(HMGB1)表达和释放的影响。方法培养HBE135-E6E7,四唑盐(MTT)法检测不同浓度IL-1β对支气管上皮细胞活力的影响,荧光定量PCR检测IL-1β刺激HBE后HMGB1mRNA表达,双抗体夹心ELISA法检测细胞上清液中HMGB1水平,蛋白免疫印迹方法检测IL-1β刺激HBE总蛋白、胞核、胞质HMGB1表达。免疫荧光观察IL-1β刺激后对HBE HMGB1的移位的影响。结果 0.1、1.0、10.0ng/mL IL-1β刺激HBE 24h对其活力无影响。IL-1β以浓度和时间依赖性方式刺激HBE的HMGB1mRNA水平升高,IL-1β增加HBE的HMGB1蛋白表达水平。在浓度依赖性实验,与对照组比较,1.0、10.0ng/mL IL-1β刺激HBE 24h后培养上清液HMGB1水平显著增加;在时间依赖性实验中,与对照组比较,10.0ng/mL IL-1β刺激HBE 12h、24h后细胞上清液中的HMGB1显著升高(P<0.05);10.0ng/mL IL-1β刺激HBE 24h后,HBE胞质蛋白明显增加,胞核蛋白减少。免疫荧光检测显示,HMGB1主要表达正常HBE的细胞核内,少量分布于细胞质,IL-1β(10.0ng/mL)刺激HBE 24h,HMGB1明显从HBE胞核向胞质转位。结论 IL-1β可以显著诱导支气管上皮细胞HMGB1的表达和主动释放,提示HMGB1可能参与了IL-1β介导慢性气道炎症过程。

Objective To investigate the effect of IL‐1βon the expression and release of high mobility group box 1 （HMGB1） from normal human bronchiolar epithelial cell （HBE） .Methods HBE135‐E6E7 was developed and methyltetrazolium （MTT） as‐say was used to assess the viability of HBE cell line under different concentration of IL‐1β.The mRNA expression of HMGB1 in HBE after stimulating with IL‐1βwere determined by Real‐time PCR;the level and location of HMGB1 in the cytoplasm ,nucleus and culture medium of HBE after stimulating with IL‐1β were detected by Western blot and ELISA assay .The expression and translocation of HMGB1 of HBE after stimulating with IL‐1β were detected by Immunofluorescence .Results 0 .1 ,1 .0 ,10 .0 ng/mL IL‐1βdid not influence the cell viability of HBE ;IL‐1β increased the mRNA expression of HMGB1 in HBE in does‐and time‐dependent manner and increased the protein expression of HMGB1 in HBE .In comparison with control group ,the levels of HMGB1 in the culture medium significantly increased after stimulation with IL‐1βat 1 .0 ,10 .0 ng/mL for 24 h（P〈0 .05）in the dose dependent experiments;and in time dependent experiments ,10 .0 ng/mL IL‐1βsignificantly increased HMGB1 level in the cul‐ture medium after stimulation for both 12 h and 24 h （P〈 0 .05） .After stimulation with IL‐1β （10 .0 ng/mL ） for 24 h ,the HMGB1 expression in cytoplasm significantly increased in plasmosin and decreased in nucleus .HMGB1 translocated from nuclei to cytoplasm after stimulation with IL‐1β（10 .0 ng/mL ） for 24 h .Conclusion IL‐1βcould induce HMGB1 expression and release in human bronchial epithelial cells ,which indicates that HMGB1 may involve in IL‐1β‐mediated chronic airway inflammation .