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目的观察机械牵张对人气道黏膜上皮细胞黏蛋白(MUC)表达的影响,并对其信号转导机制进行初步探讨。方法采用多功能小型生物撞击机构建体外培养的人气道黏膜上皮细胞牵张模型,分别给予丝裂素活化蛋白激酶(MAPK)信号通路的三条主要通路抑制剂:JNK抑制剂(SP600125)、p38蛋白激酶抑制剂(SB203580)及ERK1/2抑制剂(U0126)(浓度均为30μmol/L),并设未进行机械牵张的对照组和单纯机械牵张组。采用流式细胞仪检测牵张前后胞内游离Ca2+荧光强度的变化,激光共聚焦显微镜观察胞内游离Ca2+的分布情况,并分别采用RT-PCR和ELISA方法检测MUC5AC mRNA表达和蛋白分泌量。结果牵张能显著升高人气道黏膜上皮细胞胞内Ca2+浓度、MUC5AC mRNA和蛋白表达(均P<0.01)。U0126和SB203580能抑制牵张所致MUC5AC mRNA和蛋白表达增加(均P<0.01)。结论机械牵张能升高人气道黏膜上皮细胞黏蛋白表达,其机制可能与胞内Ca2+浓度的升高以及ERK1/2、p38 MAPK信号通路有关。
Objective To observe the effect of mechanical stretch on the expression of mucocutaneous mucin (MUC) in human airway and to explore its signal transduction mechanism. Methods Human airway mucosa epithelial cell stretch model was established by multi-functional small-scale biological impactor. Three main pathways inhibitors of MAPK signal pathway were JNK inhibitor (SP600125), p38 protein (SB203580) and ERK1 / 2 inhibitor (U0126) (30 μmol / L in concentration) were randomly divided into control group and mechanical stretch group without mechanical stretch. The changes of intracellular free Ca2 + fluorescence intensity before and after stretch were detected by flow cytometry. The distribution of intracellular free Ca2 + was observed by confocal laser scanning microscopy. The expression of MUC5AC mRNA and protein were detected by RT-PCR and ELISA respectively. Results Distraction could significantly increase intracellular Ca2 + concentration and MUC5AC mRNA and protein expression in human airway epithelial cells (all P <0.01). U0126 and SB203580 could inhibit the mRNA and protein expression of MUC5AC induced by stretch (all P <0.01). Conclusion Mechanical stretch can increase the expression of mucin in human airway epithelial cells. The mechanism may be related to the increase of intracellular Ca2 + concentration and the ERK1 / 2 and p38 MAPK signaling pathways.