摘要
目的探讨黏着斑激酶(FAK)在机械压力诱导的人气道黏蛋白(MUC)5AC分泌中的作用。方法气液相界面培养人支气管上皮NHBE细胞,用Transwell压力实验装置间断性施压于NHBE细胞,黏着斑激酶(FAK)siRNA转染NHBE细胞,Western blot法检测FAK和p-ERK1/2蛋白含量,实时荧光定量PCR检测MUC5AC mRNA表达,ELISA法检测MUC5AC蛋白相对含量。结果与空白对照组相比,机械压力能显著升高MUC5AC mRNA及蛋白含量及p-ERK1/2蛋白相对表达水平(均P<0.01)。FAK siRNA可显著降低机械压力所诱导的MUC5AC mRNA及蛋白含量的升高及p-ERK1/2蛋白表达(P<0.01),但与转染FAK siRNA对照组相比,机械压力+转染FAK siRNA组细胞表达MUC5AC mRNA及蛋白水平和p-ERK1/2蛋白相对水平仍然是增加的(P<0.05),PD98059能显著抑制机械压力所诱导的MUC5AC mRNA及蛋白表达水平的升高(P<0.01)。FAK siRNA联合AG1478作用于NHBE细胞则可显著抑制机械压力诱导的MUC5AC mRNA和蛋白表达(P<0.01)。AG1478单独作用能有效降低由机械压力所诱导的上述指标的升高(P<0.05),但与AG1478对照组相比差异仍具有显著性。结论机械压力诱导的MUC5AC高表达的信号传导通路为ERK依赖的,FAK为ERK的上游信号分子。
Objective To explore effects of focal adhesion kinase on mucin(MUC) 5AC hypersecretion induced by mechanical stress.Methods Normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface and exposed to chronic intermittent compressive mechanical stress via Transwell mechanical stress apparatus.Cells were treated with FAK siRNA.Western blot was performed to determine FAK and phosphorylation of ERK1/2 protein contents.Real-time PCR and ELISA were underwent to test MUC5AC mRNA and protein contents respectively.Results The expression level of phosphorylation of FAK at Tyr397 (p-FAK-Y397) in mechanical group was significantly higher than those in control group.Compared with the control group,mechanical stress significantly increased expressions of MUC5AC mRNA and protein contents and also the protein expression of p-ERK1/2(P < 0.01).FAK siRNA significantly attenuated the stress-induced increase in MUC5AC mRNA and protein expression and also the protein expression of p-ERK1/2 (P < 0.01).But expression of MUC5AC mRNA and protein and p-ERK1/2 protein in mechanical + FAK siRNA group was still higher than those in FAK siRNA control group(P < 0.05).PD98059 significantly decreased the stress-induced increase in MUC5AC mRNA and protein expression(P <0.01).Combination of FAK siRNA and AG1478 could significantly inhibit the stress-induced increase in MUC5AC mRNA and protein expression(P <0.01).AG1478 alone effectively decreased stressinduced MUC5AC mRNA and protein expression(P < 0.05),but when compared with AG1478 control group,the differences were still significant.Conclusions The signal transduction pathway of stress-induced overexpression of MUC5AC is ERK-dependent,FAK is one of the upstream signal molecules of ERK.
出处
《基础医学与临床》
CSCD
北大核心
2014年第5期589-594,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(31201059
31171346)
国家自然科学基金中俄国际合作项目(31211120168)
重庆市卫生局面上项目(2012-2-077)
关键词
黏着斑激酶
机械压力
黏蛋白类
细胞外信号调节激酶
focal adhesion kinase
mechanical stress
mucins
extracellular signal-regulated kinase