The Effect Of Melatonin On DNA Methylation In Human Hypertrophic Scar Fibroblast Differentiation

Rart 1 The effect of melatonin on DNA methylation in human hypertrophic scar fibroblastsPurpose To explore the effect of melatonin on DNA methylation in human hypertrophic scar fibroblastsMethods 1.Human hypertrophic scar fibroblasts of the 4^thh generations were isolated and cultured by tissue block method.The fibroblasts were identified by immunocytochemistry?ICC?.2.Cells were digested by enzymatic digestion and inoculated into 96-well plate.Melatonin groups were added with different concentrations(0,1x10^-5,1x10^-3,1mmol/L).Cell proliferation and cytotoxicity test kit?WST-1?was used to detect the proliferation activity of fibroblasts 48 hours after grouping culture.The morphological changes of fibroblasts were observed under inverted microscope.The appropriate concentration of melatonin was selected as the follow-up concentration of the experimental group.The experiment was divided into two groups:?1?control group:after cell digestion and passage,4ml FBS was added and cultured for48hours;?2?melatonin group:after cell digestion and passage,10%FBS 4ml containing 10^-3 mmol/L melatonin was added and cultured for 48 hours to collect cells;Realtime fluorescence quantitative polymerase chain reaction?RT-PCR?and Western Blot were used to detect the expression of DNMT1 gene and the content of DNMT1protein in fibroblasts between control group and melatonin group.Result 1.Morphological observation and identification of fibroblasts Human hypertrophic scar fibroblasts cultured by tissue block method were observed by inverted phase contrast microscopy.The cells grew in a whirlpool or fish-like manner.The nucleus was round-like and the cell body was flat polygonal or spindle-shaped.Observation by ICC showed that DAB staining showed that there were many cell processes,which were polygonal star-shaped or spindle-shaped,with large cell volume and brown cytoplasm.Vimentin was positive in fibroblasts.2.The effect of different melatonin concentrations on growth morphology of HSFB The effect of different concentrations of melatonin on the growth of fibroblasts were observed under inverted phase contrast microscope.Fibroblasts grew in fish colony with regular morphology under different concentrations of melatonin.Different concentrations of melatonin had no effect on the growth morphology of fibroblasts from hypertrophic scars,suggesting that melatonin had no direct toxic effect on the activity of fibroblasts.3.The effects of Melatonin Concentration on HSFB Proliferation The results of WST-1 showed that:?1?Compared with the control group,the proliferation activity of fibroblasts had no statistical significance?P>0.05?,?2?Melatonin concentration of 1x10^-3 and 1mmol/L inhibited the proliferation of human scar fibroblasts,and the proliferation activity of fibroblasts was significantly lower than that of the control group?P<0.05?.According to the experimental results,the proliferation activity of fibroblasts was significantly lower than that of the control group?P<0.05?.As shown in the results,the concentration of 10^-3 mmol/L was selected as the drug concentration of the follow-up experimental group.4.The effect of melatonin on DNMT1 expression and protein content in HSFB RT-PCR showed that the expression of DNMT1 in melatonin group was?1.86±0.094?higher than that in control group?1.00±0.082?,with significant difference?P<0.05?;Western Blot method showed that the content of DNMT1 protein in melatonin group was?0.20±0.066?higher than that in control group?0.12±0.062?,with significant difference?P<0.05?.Conclusion 1.Different concentrations of melatonin have no effect on the activity of human hypertrophic scar fibroblasts2.Melatonin inhibits the proliferation of human hypertrophic scar fibroblasts.3.Melatonin can increase the expression of DNMT1 mRNA and protein in human hypertrophic scar fibroblasts,suggesting that melatonin can increase the DNA methylation level of human hypertrophic scar fibroblasts.Rart2 Effect of melatonin on the differentiation of human hypertrophic scar fibroblasts after demethylationPurpose To investigate the effect of melatonin on the differentiation of human hypertrophic scar fibroblasts after demethylation.Methods Eight cases of human hypertrophic scar tissue were collected,and the 3rd to 6th generation of hypertrophic scar fibroblasts were isolated and cultured by tissue block method.The experiment was divided into four groups:?1?Simple medium group;?2?melatonin group(melatonin concentration is 1 x10^-3mmol/L);?3?Melatonin+Decitabine?DAC?group;?4?DAC group;after 48 hours of culture,the content of?-SMA protein in each group was detected by Western Blot.Result The results of Western Blot showed that the?-SMA content of melatonin group was 0.63±0.148;the?-SMA content of melatonin+DAC group was 0.44±0.267;the?-SMA content of DAC group was 0.48±0.167,which was lower than the simple medium group?0.87±0.089??-SMA protein level,the difference was statistically significant?P<0.05?.There was no significant difference between melatonin group and melatonin+DAC group?P>0.05?.There was no significant difference between melatonin+DAC group and DAC group?P>0.05?.Conclusion 1.Melatonin can reduce the expression of ?-SMA protein in hypertrophic scar fibroblasts;2.Methylase inhibitor?DAC?can reduce the expression of?-SMA protein in hypertrophic scar fibroblasts.