摘要
目的:探讨皮层肌动蛋白结合肽(Cortactin)对剪应力(SS)诱导的气道黏液高分泌的影响及其关键靶点。方法:体外培养气道上皮细胞Beas-2B,并随机分为6组:空白对照组、SS组、SS+Wild-Cortactin组、SS+pcDNA3-Cortactin 421 -MU组、SS+pcDNA3-Cortactin 466 -MU组、SS+pcDNA3-Cortactin 482 -MU组。用Western blot法检测Cortactin蛋白、p-Cortactin蛋白表达水平,RT-PCR、ELISA法分别检测MUC5AC 转录水平和MUC5AC的分泌量。结果:转染Cortactin各型重组表达载体后细胞Cortactin蛋白明显增加,提示转染成功。转染Wild-Cortactin后再予剪应力处理,细胞的p-Cortactin蛋白、MUC5AC mRNA及蛋白表达显著增加。而转染pcDNA3-Cortactin 466 -MU后,细胞p-Cortactin蛋白、细胞培养上清液MUC5AC的相对分泌量显著下降,但MUC5AC的转录水平无明显影响。结论:在剪应力刺激诱导的气道上皮细胞MUC5AC高分泌过程中,Cortactin具有重要的介导作用,且Y466为其介导该过程的潜在关键作用靶点。
Objective: To explore the effects and pivotal site of Cortactin on secretion of mucin(MUC)5AC induced by the shear stress(SS)in airway epithelial cells. Methods: The airway epithelial cells(Beas-2B)were cultured in vitro,and divided into 6 groups:Blank control group,SS group,SS+Wild-Cortactin group,SS+pcDNA3-Cortactin 421 -MU group,SS+pcDNA3-Cortactin 466 -MU group,SS+pcDNA3-Cortactin 482 -MU group.The relative content of Cortactin and phosphorylated Cortactin(p-Cortactin)protein was detected with Western blot,the content of MUC5AC protein and transcription level of MUC5AC were observed by ELISA and RT-PCR respectively. Results: After transfecting recombinant expression vectors of Cortactin,the levels of Cortactin were increased significantly,Which confirm Beas-2B cells were transfected with each type of Cortactin successfully.After transfecting recombinant expression vectors of Wild-Cortactin,and then stimulated with shear stress(SS),the contents of p-Cortactin,MUC5AC mRNA and MUC5AC protein were evidently higher.Whereas transfecting pcDNA3-Cortactin 466 -MU,the relative contents of p-Cortactin and MUC5AC protein were obviously decreased,however,there was no significant effect in the transcriptional level of MUC5AC mRNA. Conclusion: Cortactin plays a critical role in the process of MUC5AC hypersecretion induced by shear stress(SS) in airway epithelial cells,and the phosphorylation site Y466 of Cortactin may be a potential key target for the process.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第11期1354-1357,共4页
Chinese Journal of Immunology
基金
国家自然科学基金项目(81660010,81611530713,81811530063)
