转化生长因子-β1对慢性鼻-鼻窦炎伴鼻息肉上皮紧密连接屏障功能的影响

摘要

目的 本研究旨在探讨中国汉族人群中慢性鼻窦炎伴鼻息肉(chronic rhinosinusitis with nasal polyps,CRSwNP)患者鼻腔上皮紧密连接(tight junction,TJ)屏障功能的改变以及转化生长因子-β1(transforming growth factor-β1,TGF-β1)对其调控作用.方法 气液界面方法体外培养鼻息肉患者及健康对照鼻腔上皮细胞,待细胞分化完成后加入不同浓度的TGF-β1刺激上皮细胞72 h,检测上皮细胞跨膜阻力(transepithelial resistance,TER)及细胞间荧光素通透性的变化,评价上皮细胞屏障功能的改变;此外,分别应用实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)和免疫荧光技术检测紧密连接相关基因mRNA及蛋白表达的改变.结果 与健康对照上皮相比,鼻息肉来源的上皮细胞TER明显降低,上皮细胞对荧光素FITC-葡聚糖(FITC-Dextran)的通透性增加,10 ng/mL TGF-β1刺激可明显降低两种上皮细胞的TER,同时增加荧光素通透性.细胞紧密连接蛋白occludin及zonula occludens(ZO)-1免疫荧光结果显示,健康对照上皮细胞间紧密连接蛋白呈现连续的线性结构,而鼻息肉上皮紧密连接蛋白表达模式呈现明显破坏,包括紧密连接线性结构分离、断裂等,而经10 ng/mL TGF-β1刺激后,无论健康对照还是鼻息肉来源的上皮细胞间紧密连接蛋白表达模式均有明显破坏.结论 中国汉族鼻息肉患者的鼻腔上皮紧密连接屏障功能有明显的缺陷,而TGF-β1在诱发鼻息肉上皮屏障功能缺陷中发挥了重要的调控作用,或可作为鼻息肉治疗潜在的靶位点.%Objective To investigate the epithelial tight junction (TJ) barrier integrity changes and its regulation by transforming growth factor(TGF)-β1 in Chinese patients with chronic rhinosinusitis with nasal polyps (CRSwNP).Methods Air-liquid interface (ALI) cultures were established for human nasal epithelial cells from CRSwNP patients and healthy control subjects.Differentiated cells were stimulated with different concentrations of TGF-β1,transepithelial resistance(TER) and permeability of FITC-Dextran was measured over time.Additionally,TJ protein expression was examined by using immunofluorescence and TJ mRNA expression was determined by real-time PCR.Results In comparison with control subjects,epithelial TER decreased and FITC-Dextran permeability increased in CRSwNP-derived epithelial cells along with an irregular,patchy,and decreased expression of the TJ proteins occludin and zonula occludens(ZO)-1.10 ng/mL TGF-β1 stimulation decreased epithelial TER,increased paracellular permeability,as well as disrupted TJ protein occludin and ZO-1 expression pattern in both control and CRSwNP-derived epithelial cells.Conclusion Epithelial TJ barrier integrity was disrupted in Chinese CRSwNP patients.TGF-β1 played an important role in inducing TJ barrier defects in nasal epithelium,suggesting that it might serve as a potential therapeutic target for CRSwNP treatment.