| The change of epigenetic marks is an important regulatory factor to determine the stem cell fate and differentiation.In the past few years,the research about epigenetic regulation of stem cell biology has mainly focused on embryonic stem cells.However,the understanding on epigenetic regulation in adult stem cells is limited.Mesenchymal stem cells are the most widely studied adult stem cell population,but the konwledge about the molecular mechanisms involving in the regulation of the undifferentiated state and properties of mesenchymal stem cells remains rudimentary.Due to their merit of easy access and abundant source,adipose-derived stem cells have been the preferred material of theoretical research and clinical application in recent years.In this research,adipose-derived stem cells of Arbas Cashmere goat were used as the research object,epigenetic agents 5-azacytidine and decitabine(5-aza-2’-deoxycytidine,5-Aza-dC)were applied to change epigenetic modification of adipose-derived stem cells in vitro,and the effects of DNA methylation on regulation of the characteristics and function of adipose-derived stem cells were observed,so that to enrich correlative theory of the epigenetic regulation of stem cell differentiation and provide experimental basis for understanding the molecular mechanism of stem cells characteristics and function.1.The effects of epigenetic reagents on growth characteristics of Arbas Cashmere goat adipose-derived stem cellsAdipose-derived stem cells were cultured with the half inhibitory concentration for 24h.Through a combination of cell counting,MTT assay,and detection of cell cycle and apoptosis by flow cytometry,the results showed that the number of apoptotic cells was lest,cell viability was highest,while cell cycle was arrested in G0/G1.2.Detection of epigenetic agents on Arbas Cashmere goat adipose-derived stem cells demethylationThe gADSCs were treated by 5-Aza and 5-Aza-dC with the half inhibitory concentration for 24h,and the genome was extracted for detection.The results showed DNA methylation level was decreased,but the hydroxylmethylation level was increased.The effects of 5-Aza and 5-Aza-dC on gene transcription level were consistent,DNMT1 and DNMT3B gene transcription were inhibited and DNMT3A,TET1,TET2,and TET3 gene transcription were increased.DNMT1 expression level was decreased and TET1 and TET3 expression levels were increased by 5-Aza treatment,while TET1,TET2,and TET3 expression were increased after treatment with 5-Aza-dC,although 5-Aza-dC did not inhibit DNMT1 expression.The results indicated that 5-Aza and 5-Aza-dC induced TET family promoted 5-methylcytocine conversion into 5-hydroxymethylcytosine and DNA demethylation occurred in genome largely although the function mechanism of 5-Aza and 5-Aza-dC was slightly different.3.The effects of DNA demethylation on cell proliferation,apoptosis,and pluripotency related genesTranscription and expression of proliferation related genes TERT and PCNA,apoptosis related genes P53 and BAX,and pluripotency related genes Nanog,Oct4,and Sox2 before and after 5-Aza and 5-Aza-dC treatment were detected by immunocytochemistry,real-time quantitative PCR,and western blot.The results showed that the transcriptional level of Oct4,Sox2,TERT,PCNA,and P53 were decreased after 5-Aza and 5-Aza-dC treatment.The transcription level of Nanog was increased and that of BAX was decreased after 5-Aza treatment,while the transcription level of Nanog was decreased and that of BAX was increased after 5-Aza-dC treatment.Furthermore,the gene expression of Sox2 and BAX were improved,PCNA was decreased,and Nanog,Oct4,TERT,and P53 were constant after 5-Aza and 5-Aza-dC treatment.The results suggested that the genomic demethylation changed the transcription levels of proliferation,apoptosis,and pluripotency related genes.The improvement of stem cell pluripotency depended on the increase of Sox2 expression.4.The effects of DNA demethylation on trigerminal lineages differentiation of gADSCsThe results of detection of lipid droplets amount in adipocytes and the transcription levels of adipocyte specific factors PPARG,Adipod,Fabp4,and Leptin before and after treatment showed that lipid droplets amount was increased,PPARG transcription was decreased,and Adipod,Fabp4,and Leptin transcription were increased.NGF was detected by ELISA in neural cells before and after 5-Aza and 5-Aza-dC treatment and the content was increased.Real-time quantitative PCR restlts showed that EN02 and RBFOX3 transcription levels were elevated in the differentiated neural cells after treatment.The content of ALB and urea and AFP transcription in hepatocytes before and after 5-Aza and 5-Aza-dC treatment were detected.The results showed the content of ALB and urea and AFP expression were increased.These results indicated that 5-Aza and 5-Aza-dC treatment promoted the differentiation of gADSCs into adipocytes,neural cells,and hepatocytes.5.The effects of DNA demethylation on methylation levels of PPARG,RBFOX3,and HNF4A promoterReal-time quantitative PCR analysis demonstrated that 5-Aza and 5-Aza-dC changed transcription levels of transcription factors PPARG,RBOXF3,and HNF4A before differentiation,which were related to adipogenic,neural and hepatocyte differentiation.The methylation levels of PPARG,RBOXF3,and HNF4A promoter were detected by bisulfite sequencing.81st CpG demethylation site of PPARG promoter region,20th,44th,70th,174th,and 181st CpG methylation sites of RBFOX3 promoter region,and 4 demethylation sites of HNF4A promoter region played crucial roles in transcriptional regulation of PPARG,RBFOX3,and HNF4A.This research concluded that 5-Aza and 5-Aza-dC caused high expression of TET family to promote 5-methylcytocine conversion into 5-hydroxymethylcytosine and genomic DNA demethylation.DNA demethylation changed the transcription levels of different genes,but promoted stem cell characteristics of gADSCs dependent on Sox2 transcriptional network.Genome demethylation changed partial CpG sites methylation status in the promoter region of adipogenic,neural,and hepatocyte differentiation related transcription factors PPARG,RBOXF3,and HNF4A.Demethylation promoted gADSCs differentiation into adipocytes,nerve cells,and hepatocytes in vitro. |
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