摘要
目的探讨屋尘螨(HDM)对肌动蛋白应力纤维(F-actin)重新排布的影响及表皮生长因子受体(EGFR)相关信号通路在其中的作用。方法选取正常人支气管上皮细胞系16HBE细胞为研究对象,以HDM刺激细胞,予EGFR抑制剂AG-1478进行预处理,实验分为4组:Control组,AG-1478组,HDM组,AG-1478+HDM组,应用Western blotting检测HDM对磷酸化(p-)EGFR、F-actin及粘附连接蛋白E-cadherin和β-catenin表达的影响,应用免疫荧光技术观察F-actin、E-cadherin及β-catenin的分布变化,并测量16HBE细胞层的跨上皮电阻(TER)值和右旋糖苷(FITC-DX)的透过率。结果 HDM刺激细胞10 min时,p-EGFR表达明显增多(P<0.05),E-cadherin(P>0.05)及β-catenin(P>0.05)蛋白表达无明显改变,免疫荧光示HDM刺激后E-cadherin及β-catenin均发生分布异常,由胞膜向胞浆弥散。HDM组TER值(70.00±4.33)%显著下降,FITC-DX透过率(115.98±4.34)%明显增加,加入EGFR抑制剂后E-cadherin和β-catenin的分布异常及TER值(90.00±3.75)%、FITC-DX(101.10±2.10)%透过率均明显改善。HDM刺激后促进F-actin表达增多并出现重新排布,而EGFR抑制剂可明显抑制这一过程(P<0.05)。结论表皮生长因子受体相关信号通路参与了屋尘螨介导肌动蛋白应力纤维的重新排布,并导致气道上皮屏障破坏。
Objective To investigate the role of epidermal growth factor receptor （EGFR） signaling pathway in bronchial epithelial actin stress fiber （F-actin） rearrangement induced by house dust mite （HDM）. Methods Normal human bronchial epithelial cells （16HBE） were stimulated with HDM with or without pretreatment with AG-1478, an EGFR inhibitor. The levels of phospho（p）-EGFR, F-actin, E-cadherin and β-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and β-catenin in the bronchial epithelial cells were determined with immunofluorescence assay, and the transmembrane electrical resistance （TER） and FITC-dextran flux （FITC-DX） in the cells were measured to assess the barrier function of the bronchial epithelia. Results HDM stimulation of the cells for 10 min resulted in significantly increased p-EGFR expression （P〈0.05） without causing obvious changes in the expression of E-cadherin （P〉0.05） orβ-catenin （P〉0.05）. Immunofluorescence assay revealed delocalization of E-cadherin and β-catenin in HDM-treated 16HBE cells, shown by their diffusion from the cell membrane to the cytoplasm. In HDM-treated cells, the TER was significantly decreased to （70.00＆#177;4.33）%and the FITC-DX was significantly increased to （115.98＆#177;4.34）%;Inhibition of EGFR reversed the delocalization of E-cadherin and β-catenin, improved the TER to （90.00 ＆#177; 3.75）% and lowered the FITC-DX to （101.10 ＆#177; 2.10）%. HDM induced increased expression and rearrangement of F-actin, which was obviously inhibited by pretreatment of the cells with AG-1478 （P〈0.05）. Conclusion EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2017年第6期737-743,共7页
Journal of Southern Medical University
基金
国家自然科学基金(81670026,81500023,81470228,81270087,81270089)
国家重点基础研究发展计划“973”计划(2012CB518203)
2016年“精准医学研究”重点专项(2016YFC0905800)
广东省科技计划项目(2015A030313236,2014A030310325)~~
