DNA Methylation Levels Of Rats’Genome In Cfb Transdifferentiation Induced By Free Silica

Transdifferentiation is a specific physiological or pathological process in which differentiated cell lost its original phenotype characteristics and gives way to other types of cell due to the effect of some factors.In the process of silicosis happened,alveolar macrophage stimulated by free silica can secrete several active factors,especially TGF-β1,which can induce the transdifferentiation of lung fibroblast into myofibroblast and a serious of other cascade reactions such as the up-expression of α-SMA and collagen,the suppression of cell apoptosis and the promotion of cell proliferation,all these eventually arouse the occurrence of pulmonary fibrosis.Numerous of gene and protein’abnormal expressions are involved in fibrocyte transdifferentiation,and its mechanisms are still unclear under researches of traditional genetics.Compare with traditional genetics,epigenetic regulation has its advantages,its changes are common and reversible,which supply a target to reverse transdifferentiation.For all these reasons,the epigenetic studies on fibrocytes transdifferentiation may be very pregnant in silicosis.ObjectivePulmonary fibrosis model in vitro was established to investigate the alterations of DNA methylation patterns in fibrocytes transdifferentiation at a genome-wide level and detect the expression of DNMTs at gene and protein levels.The purpose was to supply epigenetic experimental basis for pulmonary fibrosis study and provide new ideas for clinical treatment about pneumoconiosis.Methods1.The establish of ThinCertTM model in vitro.Collected fibrocytes through lungs trypsinization and macrophages from peritoneal lavage fluid,then cocultivated them with ThinCertTM model and indirect method respectively with DMEM containing10%FBS in a humidified atmosphere of5%CO2at37℃.2.Identification for LF transdifferentiation.Macrophages were treated in duplicate with free silica in different concentrations(25μg/ml,50pg/ml and100μg/ml).Real-time PCR was performed to detect the mRNA level of a-SMA, COL Ⅰ and COL Ⅲ in LF,ELISA method was used to detect the protein expression of α-SMA,COL Ⅰ and COL Ⅲ in LF.3.The detection of DNA methylation in LF.Set0μg/ml free silica group as control,experimental groups were treated with free silica of different concentrations(25μg/ml,50μg/ml,100μg/ml),and for DAC group,the fibrocytes were pretreated with3μmol/L DAC for24h before100μg/ml free silica treatment.Cocultivate them for another24h.HPLC was used to determine the content of dC and5-dmC in genome DNA.4.Detections for mRNA and protein expression of DNMTs.Real-time PCR was used to detect mRNA levels of DNMT1,DNMT3a,DNMT3b,MBD2,MeCP2and the protein expression of DNMT1,DNMT3a,DNMT3b,MBD2,MeCP2was determined by ELISA method.Results1.AMs and LFs collected from SD rats grew well in vitro and they were suitable for further study.The cocultivation model was successfully established.2.Identification of transdifferentiation.In ThinCertTM,the expression of α-SMA,COL Ⅰ and COL Ⅲ in experimental groups were higher than control group(P<0.05),and the higher the free silica concentration,the more the expression of α-SMA,COL Ⅰ and COL Ⅲ.The expression of α-SMA,COL Ⅰ and COL Ⅲ in ThinCertTM were all higher than that in indirect model.3.The content of dC and5-dmC in genome DNA.Compared with control group(5.53%±0.044%),the methylation levels of experimental groups[25μg/ml(4.30%±0.067%),50μg/ml(3.86%±0.062%),100μg/ml(3.50%±0.036%)]were decreased by22.2%,30.2%,36.7%respectively,and the differences were statistically significant(P<0.05).The methylation level of genome DNA in DAC group was dropped by20.6%compared with100μg/ml group,and the difference was statistically significant(P<0.05).4.The expression of mRNA and protein of DNMTs.The difference of DNMT1mRNA between control group and25μg/ml groups were not statistically significant(P>0.05);but DNMT1mRNA in50μg/ml group,100μg/ml group and DAC group was dropped by27.6%,42.1%and64.4%respectively compared with control group, and the differences were statistically significant(P<0.05).The mRNA expression of DNMT3a in25μg/ml group,50μg/ml group,100μg/ml group and DAC group was decreased by14.5%,29.5%,38.5%and52.2%respectively compared with the control group,and the differences were statistically significant(P<0.05).Compared with control group,the mRNA expression of DNMT3b in DAC group drop35.5%,and the difference was statistically significant(P<0.05).mRNA expression in other experimental groups were not obviously decreased,and the differences had no statistically significances(P>0.05).Compared with control group,the mRNA level of MBD2in25μg/ml group and50μg/ml group had no significant decreasing(P>0.05);and the levels in100μg/ml group and DAC group was dropped by14.5%and56.8%respectively,the differences were statistically significant(P<0.05).The mRNA level of MeCP2in DAC group reduced71.5%compared with control group,and the difference was statistically significant(P<0.05);The decrease in other experimental groups were not obvious,the differences were not statistically significant(P>0.05).The expression of protein was consistent with mRNA expression,the protein expression of DNMT1,DNMT3a and MBD2in experimental groups all reduced compared with control group,and the expression of DAC group was the lowest.The decrease of DNMT3b and MeCP2expression were not obvious.Conclusion1.ThinCertTM model to cocultivate macrophage and fibrocytes are established successfully,and it can be used in the study of fibrocytes transdifferentiation induced by free silica.2.Free silica can result in a dose-dependent reduction of DNA methylation at the mercy of DNMT1、DNMT3a and MBD2in ThinCertTM model.