The Mediated Dffect Of Cortactin In Airway Mucin Secretion Induced By Shear Stress

Objective:To explore the effects of Cortactin on secretion of mucin（MUC）5AC induced by the shear stress（SS）in airway epithelial cells,and investigate the pivotal site of Cortactin in this mediated process.Methods:1、The human airway epithelial cells（HBE16）were cultured in vitro,and divided into 6 groups.Group A:Blank control groupGroup B:SS groupGroup C:SS+Wild-Cortactin groupGroup D:SS+pcDNA3-Cortactin ⁴²¹-MU groupGroup E:SS+pcDNA3-Cortactin ⁴⁶⁶-MU groupGroup F:SS+pcDNA3-Cortactin ⁴⁸²-MU group2、Recombinant expression vectors wild-Cortactin and each mutant type of Cortactin(pcDNA3-Cortactin ⁴²¹、pcDNA3-Cortactin ⁴⁶⁶-MU、pcDNA3-Cortactin⁴⁸²-MU)were transfected into HBE16 cells respectively.The protein levels of Cortactin in each group were determine by Western blot,Only after the recombinant expression vectors were transfected into cells successfully,can we continue to implement the following-up experiments.3、Using the shear stress（SS）from rotary device as the stimulating factors,in order to establish the model of epithelial hypersecretion,the operating parameters was Set to 30r/min,30min/time.4、The relative content of phosphorylated Cortactin（p-Cortactin）protein in each group was detected with Western blot,the levels of MUC5AC proteinin supernate and MUC5AC mRNA of lysate cell solution were observed by ELISA and RT-PCR respectively.After the HBE16 cells were transfected with each type of Cortactin recombinant expression vectors and stimulated by SS,we can infer the key fuction site of Cortactin by comparing the p-Cortactin and MUC5AC protein conten in supernate difference between each group.Results:1、After transfecting recombinant expression vectors wild-Cortactin and each mutant type of Cortactin,Compared with blank control group,the levels of Cortactin in transfected groups were increased significantly（p<0.01）,Which confirm HBE16cells were transfected with each type of Cortactin successfully.2、Compared with blank control group,the contents of p-Cortactin,MUC5AC mRNA and MUC5AC protein in SS group were evidently higher（p<0.01）.3、The relative content of p-Cortactin and MUC5AC inSS+Wild-Cortactin group were risen prominently compared with SS group（p<0.01,p<0.05）.4、Compared with SS+Wild-Cortactin group,the relative contents of p-Cortactin and MUC5AC protein in cell supernate in SS+pcDNA3-Cortactin ⁴⁶⁶-MU group were obviously decreased（p<0.01）,however,there was no significant effect in the transcriptional level of MUC5AC mRNA（p>0.05）,whereas transfection with the pcDNA3-Cortactin⁴2121 or pcDNA3-Cortactin⁴8282 had no effect on MCU5AC mRNA transcription and MUC5AC protein secretion（p>0.05）.Conclusion:Cortactin plays a critical role in the process of MUC5AC hypersecretion induced by shear stress（SS）in airway epithelial cells,mutating the phosphorylation site Y466of Cortactin may inhibite hypersecretion of MUC5AC protein obviously induced by shear stress（SS）.