The Influence Of CXXC5 Expression To The Differentiation Of MLFs And The CD40/CD40L Signal Pathway

Objective:To clarify the effects of CXXC5 on the Mouse Lung Fibroblasts(MLFs).To clarify the effects of CXXC5 in the process of transdifferentiation which occurred in the MLFs and CD40/CD40 L signaling pathway.After the TGF-β1 stimulus,adjust the expression of CXXC5,to clarify the role of CXXC5 and TGF-β1 in MLFs differentiation and CD40/CD40 L signaling pathway.Methods:Construction of adenovirus vector: the adenoviral vectors containing the green fluorescent protein(Ad-GFP),the adenoviral vectors containing the green fluorescent protein and the target gene CXXC5(Ad-CXXC5),the adenoviral vectors containing the green fluorescent protein and the specific shRNA CXXC5(Ad-shR-CXXC5).MLFs were divided into 8 groups.Tissue adherence method to culture MLFs.Adenovirus infects logarithmic growth cells.explore the best MOI value.CCK-8 and flow cytometry methods detect each group of proliferation activity and apoptosis rate.ELISA detect the expression of Col1 aI,Col1aIII,sCD40 L.RT-qPCR detect the expression of CXXC5,α-SMA,proCol1 aI,proCol1a III,α-SMA,CD40 and CD40 L.Western blot was used to evaluate the protein of CXXC5,α-SMA,CD40 and CD40 L.Results:Adenovirus vector construceed Successfully.The best MOI value for the three viruses at 48 hours of infection was 100.The cell proliferation activity can be measured through CCK-8:Overexpression of CXXC5 could inhabit MLFs cell proliferation activity(P < 0.05);down-regulation of CXXC5 could promote MLFs cell proliferation activity(P<0.05);After joining TGF-β1 stimulation,MLFs cell proliferation activity was promoted;overexpression of CXXC5 could inhabit the role of TGF-β1;and down-regulation of CXXC5 could promote the role of TGF-β1.Apoptosis rate can be detected in each group by flow cytometry.Overexpression of CXXC5 could enhance the apoptosis of MLFs(P < 0.05),and down-regulation of CXXC5 could inhabit MLFs cell apoptosis(P <0.05);After joining TGF-β1 stimulation,MLFs cell apoptosis was inhabited;overexpression of CXXC5 could inhabit the role of TGF-β1;and down-regulation of CXXC5 could promote the role of TGF-β1.Test results of ELISA.Overexpression of CXXC5 can reduce expression of theCol1 aI,Col1aIII,sCD40 Land α-SMA.Silencing CXXC5 down regulate the level of α-SMA CollaI,Col1 aIII and sCD40 L.Results of RT-qPCR..Overexpression of CXXC5 can reduce the proCol1 aI,proCollaI,sCD40 L andα-SMA mRNA.Silencing CXXC5 up regulate the level of α-SMA and CollaI mRNA.After joining TGF-β1 stimulation,these mRNA were up regulated.TGF-β1 reduce CXXC5,proCol1 aI,proCol1aIII,sCD40 L and α-SMA mRNA.Results of Western blot:Overexpression of CXXC5 could up regulate the expression levels of CXXC5 proteins and down regulate the α-SMA,CD40 and CD40 L proteins.Conclusions:(1)CXXC5 can promote apoptosis and inhibit proliferation of MLFs cells.(2)Overexpression of CXXC5 can inhibit the transdifferentiation of fibroblasts into myofibroblasts.(3)CXXC5 can inhibit the effect of TGF-β1 on pulmonary fibrosis transdifferentiation in mice.(4)CXXC5 can regulate the formation of pulmonary fibrosis by regulating the CD40/CD40 L signaling pathway through negative feedback.