The Study Of Effection Of DAPT In Wnt Signal Pathway Of Pulmonary Fibroblast Phenotype Transdifferentiation

Objective：To study the expression variability of wnt1，beta-catenin and myofibroblastcell marker factor named alpha-SMA in Wnt signal pathway’s after gamma-secretaseinhibitor of DAPT blocks Notch signaling pathway. To explore the indirect or directinteractions between the signaling pathways of Wnt/beta-catenin and Notch, and tostudy the expression of Wnt/beta-catenin signaling pathway of DAPT’ inhibition oflung fibroblast cell phenotypic transdifferentiation.Methods：(1) To get the lung tissue from SD rats of2or3-day-old. Isolating and cμlturinglung fibroblast by mean of the method of trypsin digestion, With Vimentinimmunocytochemistry, testing purity of the third generation of lung fibroblast;(2)Then use fetal bovine serum-free in DMEM medium synchronous cμlture cell for24h, and then we devided them into control group, TGF-β1group, TGF-β1+DAPTgroup;(3) Respectively in the TGF-β1group and TGF-β1+DAPT group to add5ng/ml TGF-β1induced fibroblast into myofibroblast, and TGF-β1+DAPT group toadd500nmol/L DAPT inhibiting fiber cell proliferation, and24h for lung fibroblastsand the morphological change was observed;(4) We test the expression variability ofα-SMA, wnt1, β-catenin mRNA by RT-PCR method and the expression variability ofβ-catenin protein by Western-blot method;(5) SPSS13.0statistic software to do theanalysis, also using t-test to compare the average value of two samples and applyingone-factor analysis of variance to mμlti-sample comparisons and having LSD-t testfor inter-groups comparisons.Resμlts：(1) Detection of Vimentin immunocytochemistry: brown particles are visible inalmost of cytoplasmic, which prove the use of trypsin digestion to the cells of thirdgeneration are almost of lung fibroblasts;(2) α-SMA mRNA value of the expression: control group α-SMA mRNA(0.3850.015), TGF-β1group(0.615±0.025),TGF-β1+DAPT group(0.345±0.027), TGF-β1group α-SMA mRNA value issignificantly higher than the control group and TGF-β1+DAPT group, the differencewas statistically significant （p<0.05）, but control group compared withTGF-β1+DAPT group is no significant difference, that is, the difference was notstatistically significant(p>0.05);(3) wnt1mRNA value of the expression: controlgroup wnt1mRNA value(0.233±0.005), TGF-β1group(0.257±0.010),TGF-β1+DAPT group(0.209±0.016), show TGF-β1group wnt1mRNA value issignificantly higher than the control group and TGF-β1+DAPT group, the differencewas statistically significant （p<0.05）;(4) β-catenin mRNA value and proteinexpression:control group β-catenin mRNA value and protein relative absorptionspectrophotometer degree value(0.984±0.031and0.352±0.023), TGF-β1group(1.248±0.021and0.431±0.014), TGF-β1+DAPT group(0.895±0.056and0.102±0.024), show TGF-β1group is significantly higher than the control group andTGF-β1+DAPT group, the difference was statistically significant（p<0.05）.Conclusions：(1) TGF-beta1can induce the lung fibroblast transform to myofibrolasts, theexpression of α-SMA, β-catenin and wnt1were increased in the transformationprocess;(2) After DAPT blocks Notch signaling pathway, affecting theWnt/beta-catenin signaling pathway in the fibroblasts phenotypic transdifferentiation,lower the expression of α-SMA, beta-catenin, and wnt1mRNA and protein;(3) Thereis an interrelationship between Wnt/beta-catenin signaling pathway and Notchsignaling pathway.