Study On The Effect Of Thy-1 DNA Methylation In Pulmonary Fibrosis By Coal Dust

Objective Using indirect cultured experiment method,to observed the expression of COL-I,COL-III,?-SMA,Thy-1,Thy-1 DNA methylation,DNMT1 and DNMT3a/b in human embryonic lung fibroblasts,which cause fibrosis by coal dust and silica dust through THP-1 derived macrophages in vitro.To study whether Thy-1 DNA methylation changes in human embryonic lung fibroblasts fibrosis by coal dust and silica dust.Methods Using CCK-8 colorimetric method to determine expousure dose of THP-1 derived macrophages induced by coal dust and silica dust. We starting from THP-1 monocytes differentiated into macrophages using PMA(Phorbol 12-myristate 13-acetate) culture 24 hours, collectted suspension of 100 μg/ml coal dust and silica dust to stimulate THP-1 derived macrophages 24 hours,and then to stimulate human embryonic lung fibroblasts 24 h,48h,72 h.We will proceeding experiments:1.THP-1 derived macrophages IL–6 and TGF-?1 in the supernatant fluid, human embryonic lung fibroblasts TGF-?1,COL-I,COL-III,?-SMA protain containing in the supernatant determined by enzyme-linked immunoassay(ELISA).2.Real-time fluorescent quantitative PCR to detect COL-I,COL-III,?-SMA,Thy-1,DNMT1,DNMT3 a and DNMT3 b mRNA expression in human embryonic lung fibroblasts.3. Nest landing methylation specific PCR to detect Thy-1 DNA methylation.Results(1) THP-1 derived macrophages exposed to different concentrations(0, 25, 50, 100, 150, 200 μg/mL), There were significantly differences compared with control group in 150, 200 μg/mL, cell activity were reduced gradually(P﹤0.05).(2) The high expression of IL-6 and TGF-?1 compared with control group have significantly difference in Macrophages by coal dust and silica dust(100μg/mL)(P﹤0.05).(3)Human embryonic lung fibroblasts exposed to supernatant fluid from macrophages 24 h,48h and 72 h by coal dust and silica dust, TGF-?1,COL-I,COL-III, ?-SMA(mRNA and protein)were significantly higher than control group,and as exposed time increased,the expression increased gradually(P﹤0.05).The expression of 72 h Coal dust group compared with 48 h decreased,there was no significantly difference.(4)The expression of Thy-1 compared with control group have significantly decrease in Macrophages by coal dust and silica dust(24h,48 h,72h)(100μg/mL)(P﹤0.05).(5) The level of Thy-1 DNA methylation compared with control group have significantly increase in Macrophages by coal dust and silica dust(24h,48 h,72h)(100μg/mL)(P﹤0.05).(6) Human embryonic lung fibroblasts exposed to supernatant fluid from macrophages 24 h,48h and 72 h by coal dust and silica dust, the expression of DNMT1、3a、3b increased gradually(P﹤0.05), DNMT3 a of coal dust group(24h) with DNMT3 b of silica dust group(48h) compared with control group was reduced, there was no significant difference.Conclusions(1)IL-6 and TGF-?1 were higher expression in macrophages exposed by coal dust and silica dust.(2)IL-6 and TGF-?1 in macrophages can activate TGF-?1 in MRC-5.(3)The activitated TGF-?1 promoted human embryonic lung fibroblasts fibrosis.(4)With MRC-5 cells fibrosis changes, Thy-1 expression were reduced gradually.(5)In the role of DNA methylation transferase,Thy-1 DNA sequences were hyper-methylation.