Effects Of Wnt/?-catenin Signaling Of Alveolar Epithelial Cells On Interstitial Properties Of Pulmonary Fibroblasts

Objective On the basis of establishing the co-culture model of human alveolar epithelial cells(A549)and human pulmonary fibroblasts(IMR-90)in vitro,we explored the molecular mechanism that effects of Wnt/?-catenin signaling in the alveolar epithelial cells on the interstitial properties of pulmonary fibroblasts,to provide a theoretical basis for the clinical treatment of idiopathic pulmonary fibrosis.Methods 1.A model of air-liquid interface culture of human alveolar epithelial cells A549was established in vitro.We wsed hematoxylin and eosin histochemical staining(HE??),scanning electron microscope and transmission electron microscope to detect the surface morphology and internal ultrastructure of human alveolar epithelial cells A549 that cultured in airliquid interface.A549 cells of alveolar epithelium were cultured in air-liquid interface(ALI),and the three-dimensional cell culture model of A549 cells was established.To identify this model from morphology and molecular aspects.At the same time,we isolated the A549 cells that cultured in ALI for 3 days,1 week,2 weeks and 3 weeks,to analysis the SP-C,AQP-5,TTF-1,ZO-1 and MUC5B molecular expression by western blot,immunofluorescence staining,and RT-PCR.2.Coculturing A549 cells that cultured in ALI above and pulmonary fibroblasts,used Wnt3 a protein and XAV939 to alter the Wnt/?-catenin signaling in epithelial cells,also treated the coculture model with BLM.To explore the relationship between Wnt/?-catenin signaling and BLM.Last,we used Western Blot to test the cell marker vimentin of pulmonary fibroblasts and ?-SMA of myofibroblast in the co-culture model,moreover,used ELISA to analyze the secretion of inflammatory cytokines IL-1?,IL-6 and TF-? in coculture medium.Results 1.The A549 cells cultured in ALI formed a complete cells layer on the ALI cultured membrane.Under the electron microscope,the cell morphology is irregular,the surface is convex,and there is a large amount of secretion in the cells clearance.The Outlines of individual cells and organelles were not obvious,and they were crowded together.However,the ALI cultured cells were more compacted with clear cell junctions and abundant mucus on the apical surface.As expected,the expression of AECI cell specific marker AQP-5 and AECII cell marker SP-C were observed on the cytomembrane and in the cytoplasm of subset cells,respectively.Consistently,the AECI cell marker proteins AQP5 and TTF-1 were also increased in ALI cultures compared to submerged cultures.An increased mucin 5B(MUC5B)was also observed in ALI cultures.Interestingly,the epithelial tight junction protein ZO-1 was detected in both ALI cultures and submerged cultures,except it failed to be detected in ALI cultures of 3 days.In addition,transcriptional analysis revealed that transcripts of SPC,AQP-5,TTF-1,MUC5 B and ZO-1 were detected significantly in ALI cultures compared to submerged cultures,particularly in the 2-week-old ALI cultures and P<0.05.2.On the other hand,under the co-culture of alveolar epithelium and pulmonary fibroblast,activating the Wnt/ ?-catenin signaling in epithelial cells and stimulated some groups with BLM,made the expression of vimentin protein was decreased of fibroblast surface markers,P<0.05.However,the expression of alpha-sma protein in myofibroblasts was significantly increased,and P<0.05.The expression of TGF-?RI protein increased correspondingly.In addition,the secretion of IL-1?,IL-6 and TNF-? in the co-culture medium was also significantly increased and P<0.05.Interestingly,in the experimental group that XAV939 and BLM were all treated,the expression of alpha-sma protein and TGF-beta RI protein and inflammatory factors decreased compared with the single BLM stimulation.Conclusion 1.A549 cells grown well under the ALI culturing,and formated an intact epithelial cell layer on the membrane.2.An ALI epithelial culture model of A549 cells was established,and it indicated partial alveolar epithelial cell properties,so the ALI-cultured A549 cells may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.3.In the co-culture model of alveolar epithelial cells and pulmonary fibroblasts,changed the Wnt/ ?-catenin signaling of epithelial cells affected the secretion of epithelial cells,and interstitial properties of pulmonary fibroblasts in co-culture;made the fibroblasts were characterized by fibrosis.4.In the alveolar epithelial cells and pulmonary fibroblasts co-cultured system in vitro,XAV939 attenuated the fibrotic characteristics of two cells in the co-culture system induced by BLM by inhibiting the Wnt/?-catenin signaling in the alveolar epithelial cells.