Fuction And Mechanism Of MiR 146a In The Regulation Of Fibroblast Phenotype Transdifferentiation

Objective: 1.Through induced pulmonary fibrosis model in mice and culturing mouse lung fibroblasts in vitro,after stimulating phenotype transformation by TGF-?1 to detect TRAF6 protein expression and miR146 a expression in different degrees of transformation by Western-blotting,RT-PCR this study intends to demonstrate that miR146 a is a regulation factor in lung fibroblast phenotypic transformation,exploring the mechanisms of mi R146 a in pulmonary fibrosis.Methods : 1?Divided 40 C57BL/6 mice into 2 groups randomly: control group,bleomycin-induced pulmonary fibrosis group(model group);the control group was treated with normal saline through intratracheal instillation,the model group was treated with bleomycin solution(2.5mg/kg),the mice were sacrificed at fourth weeks after building model.2?Verify whether the pulmonary fibrosis was successfully by hematoxylin-eosin staining(HE staining);3 ? Lung fibroblasts were identified by two methods,i.e.,morphological changes of the cells were observed and photographed under an inverted microscope,and fibroblast surface markers were determined by immunocytochemical staining.3.Phenotypic transformation of lung fibroblasts were stimulated with different concentrations of TGF-?1 and the expressions of ?-SMA and TRAF6 proteins were detected by Western blot and the expression of miR-146 a was determined by qRT--PCR,so as to explicit the role of miR-146 a in the phenotypic transformation of lung fibroblasts in mice.Results: The hematoxylin-eosin staining showed that the lung were change as pulmonary fibrosis in the model group,and successfully cultured Lung fibroblasts,and have induced phenotype transformation of lung fibroblasts by different concentrations of TGF-?1.1?Different concentrations of TGF1 stimulated lung fibroblast conversion phenotype,the protein expression of ?-SMA and TRAF6 have significant statistical differences between the groups(P<0.01,P<0.05 and(TRAF6)F=41.25;(a-SMA)F=150.04),with the increase of the concentration of TGF1,the expression of a-SMA protein and TRAF6 protein were increased in a concentration dependent manner;2?there was statistical significance between the four groups the gene expression of miR146a(P<0.01,P<0.05 and F=335.00).The highest expression by the stimulation of 5ug/l TGF-?1,and then showed a concentration dependent decrease.3?The higher the phenotype transformation rate of lung fibroblasts induced by TGF-?1(the higher expression of-SMA),the lower the expression of miR146 a.Conclusions:(1)TGF-beta 1 promotes the transformation of lung fibroblasts into myoblasts in a certain concentration range.(2)mi R146 a play a negative regulating effect role in lung fibroblasts transformed myoblasts.