蛋白酪氨酸激酶6对TNF-α诱导的人气道上皮屏障功能减损的影响

Effect of protein tyrosine kinase 6 on human airway epithelial barrier dsyfunction induced by TNF-α

目的:探讨蛋白酪氨酸激酶6(PTK6)对TNF-α诱导的人气道上皮屏障功能减损的影响及相关机制。方法:体外培养人气道上皮16HBE细胞,予TNF-α刺激,分别转染PTK6 siRNA和recombined PTK6,以转染Scramble siRNA和Empty vector为对照。四甲基偶氮唑盐法(MTT)检测细胞活性;跨皮细胞组抗仪检测细胞跨皮细胞阻抗(TER);辣根过氧化物酶(HRP)流量法反应细胞通透性;RT-PCR检测ZO-1、Occludin mRNA水平;Western blot检测PTK6、ZO-1、Occludin、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)、磷酸化c-Jun氨基端激酶1/2(p-JNK1/2)和磷酸化p38(p-p38)蛋白水平。结果:TNF-α刺激后,细胞ZO-1、Occludin转录及蛋白水平、TER值显著降低,细胞通透性增高,同时PTK6、p-ERK1/2、p-JNK1/2和p-p38蛋白水平显著增高(P<0.05);上调PTK6使ZO-1、Occludin转录及蛋白水平和TER值进一步降低,细胞通透性和p-JNK1/2、p-p38蛋白水平也进一步增高(P<0.05),但下调PTK6后上述指标呈相反变化(P<0.05);上调或下调PTK6对p-ERK1/2无明显影响。结论:下调PTK6可以降低JNK1/2、p38MAPK磷酸化水平,进而改善TNF-α诱导的气道上皮屏障功能减损。

Objective：To investigate the effect of protein tyrosine kinase 6（PTK6） on TNF-αinduced human airway epithelial barrier dysfunction and mechamism.Methods： After cultivating 16HBE cells in vitro,the recombined PTK6 and PTK6 siRNA was re-spectively transfected into the cells,with empty vector and scramble siRNA as control.The cells were incubated with exogenous TNF-α.Cell viability was detected by MTT assay.Cells TER and permeability were detected.ZO-1and Occludin mRNA were analyzed by RT-PCR.PTK6,ZO-1, Occludin, p-ERK1/2, p-JNK1/2 and p-p38MAPK protein were assayed by Western blot.Results： TNF-α remarkably decreased ZO-1 and Occludin mRNA and protein and TER;increased cells permeability,as well as p-ERK1/2,p-JNK1/2 and p-p38 protein（P〈0.05）.Upregulation of PTK6 further decreased ZO-1 and Occludin mRNA and protein and TER,enhanced cells permeability and p-JNK1/2 and p-p38 protein（P〈0.05）,and downregulated PTK6 ,the changes of these indices were opposite（P〈0.05）.Whereas upregulation or downregulation of PTK6had no effect on p-ERK1/2.Conclusion： Downregulation of PTK6 inhibits the phosphorylation of JNK1/2 and p38MAPK,thus improving TNF-α-induced human airway epithelial barrier dysfunction.