呼吸道合胞病毒持续感染导致人支气管上皮细胞囊性纤维化穿膜传导调节蛋白功能障碍

Respiratory syncytial virus persistent infection causes acquired CFTR dysfunction in human bronchial epithelial cells

目的:呼吸道合胞病毒(respiratory syncytial virus,RSV)持续感染可能是导致气道慢性疾病的病理原因,然而,其作用机制尚不清楚。囊性纤维化穿膜传导调节蛋白(cystic fibrosis transmembrane conductance regulator,CFTR)是一种顶端膜氯离子(Cl-)通道,对上皮液体、Cl-和碳酸氢盐运输的调节至关重要。CFTR功能障碍将导致支气管分泌物改变和黏液清除受损,与气道炎症有关。我们前期在RSV感染的小鼠模型中观察到气道上皮细胞CFTR表达下调。本研究通过构建RSV持续感染的气道上皮细胞模型,进一步研究其对CFTR表达和功能的影响。方法:以病毒感染复数(multiplicity of infection,MOI)为0.01的RSV感染16HBE14o-细胞后继续传代。以real-time RT-PCR、免疫荧光及蛋白质印迹法检测CFTR的表达,以新型氯离子荧光探针MQAE测量细胞内Cl-浓度和全细胞膜片钳记录Cl-电流。结果:16HBE14o-细胞感染RSV病毒后存活至第3代(G3)。CFTR的表达在RSV感染后从G1到G3逐渐降低,同时伴随着CFTR功能的降低。16HBE14o-细胞暴露于RSV可导致TGF-β1表达进行性升高及其下游信号分子Smad2磷酸化。用SB431542阻断TGF-β1信号通路可阻止Smad2磷酸化,增加CFTR的表达。结论:RSV感染可导致气道上皮细胞CFTR功能缺陷,其作用可能是通过激活TGF-β1信号通路介导的。

Objective:Many studies have shown that respiratory syncytial virus persistent infection may be the main cause of chronic respiratory pathology.However,the mechanism is unclear.Cystic fibrosis transmembrane conduction regulator(CFTR)is an apical membrane chloride channel,which is very important for the regulation of epithelial fluid,chloride ion,and bicarbonate transport.CFTR dysfunction will lead to changes in bronchial secretions and impair mucus clearance,which is related to airway inflammation.In our previous study,we observed the down-regulation of CFTR in airway epithelial cells in respiratory syncytial virus(RSV)infected mouse model.In this study,we further investigated the expression and function of CFTR by constructing an airway epithelial cell model of RSV persistent infection.Methods:16HBE14o-cells were infected with RSV at 0.01 multiplicity of infection(MOI).The expression of CFTR was detected by real-time RT-PCR,immunofluorescence,and Western blotting.The intracellular chloride concentration was measured by N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide(MQAE)and the chloride current was measured by whole-cell patch clamp recording.Results:16HBE14o-cells infected with RSV were survived to successive passages of the third generation(G3),while the expression and function of CFTR was progressively decreased upon RSV infection from the first generation(G1)to G3.Exposure of 16HBE14ocells to RSV led to the gradual increase of TGF-β1 as well as phosphorylation of Smad2 following progressive RSV infection.Disruption of TGF-β1 signaling by SB431542 prevented Smad2 phosphorylation and rescued the expression of CFTR.Conclusion:RSV infection can lead to defective CFTR function in airway epithelial cells,which may be mediated via activation of TGF-β1 signaling pathway.