肺泡巨噬细胞TLR4/MyD88信号通路参与并介导了机械通气所致的肺损伤  

Alveolar macrophage TLR4 / My D88 signaling pathway contributes to ventilator-induced lung injury in rats

作　　者：黄翠源 潘灵辉[1] 林飞[1] 钱卫[1] 李玮[1] 

机构地区：[1]广西医科大学附属肿瘤医院麻醉科,广西南宁530021

出　　处：《细胞与分子免疫学杂志》2015年第2期182-185,189,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81060008)

摘　　要：目的探讨肺泡巨噬细胞Toll样受体4/髓样分化因子88(TLR-4/My D88)信号通路在呼吸机相关性肺损伤中的作用。方法 30只成年SD大鼠行经口气管插管,给予40 m L/kg潮气量通气240 min,建立呼吸机相关性肺损伤模型,机械通气结束后,使用4℃PBS经气管导管缓慢注入并回收支气管肺泡灌洗液(BALF),提纯肺泡巨噬细胞。收集并培养肺泡巨噬细胞,随机分为3组:PBS刺激组(CON组);TNF-α刺激联合PBS组(STI组);TNF-α刺激联合抗TLR4单克隆抗体(m Ab)干预组(ANT组);每组8个样本。CON组细胞用PBS结合2 h后,继续用PBS培养16 h;STI组细胞用PBS结合2 h后,采用20 ng/m L TNF-α培养16 h;ANT组细胞用PBS液及TLR4 m Ab结合2 h后,用20 ng/m L TNF-α培养16 h。ELISA检测各组上清液TNF-α、IL-1β、IL-6的表达;反转录PCR检测各组肺泡巨噬细胞TLR4、TLR9、髓样分化因子88(My D88)、核因子κB(NF-κB)的mRNA表达,Western blot法检测各组肺泡巨噬细胞TLR4、TLR9、My D88、NF-κB的蛋白表达。结果与CON组比较,STI组及ANT组细胞培养上清液TNF-α、IL-1β、IL-6的浓度明显增高;STI组肺泡巨噬细胞TLR4 mRNA、My D88 mRNA、NF-κB mRNA表达水平与蛋白表达水平明显增高;ANT组肺泡巨噬细胞TLR4 mRNA、My D88 mRNA、NF-κB mRNA表达水平与蛋白表达水平无明显变化。与STI组比较,ANT组细胞培养上清液TNF-α、IL-1β、IL-6的浓度明显降低;肺泡巨噬细胞TLR4 mRNA、My D88 mRNA、NF-κB mRNA表达水平与蛋白表达水平明显下调。3组TLR9 mRNA与蛋白表达水平相近。结论炎症因子刺激可调节TLR4、My D88、NF-κB分泌增加,肺泡巨噬细胞TLR4-My D88信号通路参与并介导了机械通气所致的肺损伤。Objective To investigate the role of alveolar macrophages Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88） signaling in ventilator-induced lung injury （VILI） in rat model. Methods Thirty adult male Sprague-Dawley rats were ventilated with high tidal volume （HTV） （40 ml/kg） for 240 minutes to establish VILI model after oral intubation. Then 4℃ PBS was infused through the endotracheal tube and alveolar macrophages （AMs） were purified from the bronchoalveolar lavage fluid （BALF）. The AMs were randomly divided into 3 groups （n = 8 each）： PBS stimulating group （group CON）, TNF-α stimulating combined with PBS blocking group （STI group）, and TNF-α stimulating combined with TLR4 monoclonal antibodies （TLR4 mAb） blocking group （ANT group）. CON group was cultured for 16 hours after blocked by PBS for 2 hours. STI group was cultured with TNF-α （20 ng/mL） for 16 hours after blocked by PBS for 2 hours. ANT group was cultured with TNF-α （20 ng/mL） for 16 hours after blocked by PBS and TLR4 mAb for 2 hours. The cell culture supernatants were collected for determination of the expressions of TNF-α, IL-1β and IL-6 with ELISA. The expressions of TLR4, TLR9, MyD88 and nuctear factor κB （NF-κB） at both mRNA and protein levels were detected by reverse transcription PCR and Western blotting, respectively. Results Compared with CON group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants increased significantly in STI group and ANT; the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs rose significantly in STI group, but there was no significant difference in the mRNA and protein levels of TLR4, MyD88 and NF-κB in ANT group. Compared with STI group, concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants, the mRNA and protein levels of TLR4, MyD88 and NF-κB in AMs decreased significantly in ANT group. There was no significant difference in TLR9 mRNA and protein levels among the three groups. Conclus

关 键 词：呼吸机相关性肺损伤 肺泡巨噬细胞 TOLL样受体4 TOLL样受体9 TNF—α 

分 类 号：R392.12[医药卫生—免疫学] R563[医药卫生—基础医学]