The Differences Of Growth Characteristics And Epigenetic Modification Between Fetal Fibroblast Cells And Mesenchymal Stem Cells Of Arbas Cashmere Goat

Somatic cell nuclear transfer(SCNT)is the main technology for breeding new transgenic varieties.The advantage of SCNT is not only to increase the number of excellent varieties,protect the endangered animals effectively,but also to provide an important technology for research of cell differentiation and gene regulation.At present,fetal fibroblast is the most commonly used nuclear donor cell in SCNT.Because of good proliferation ability,fetal fibroblast has be used for cytogenetic modification,and then be used as a nuclear donor cell to produce viable transgenic offspring.However,as a nuclear donor cell,it has the disadvantages of limited passage in vitro and low efficiency of gene transfection,which will impede the cultivation on new transgenic varieties.Compared with fetal fibroblasts,stem cells in adult tissues have many advantages,such as convenient to draw materials,easy to culture,high proliferative ability and low differentiation,and has been widely applied in many fields,such as breeding new transgenic varieties,regenerative medicine and so on.In this study,Arbas Cashmere goat fetal fibroblasts(FFCs),adipose-derived stem cells(ADSCs),bone marrow stem cells(BMSCs)and muscle-derived satellite cells(MDSCs)were used as research objects,the expression of pluripotency,proliferation and apoptosis related genes were compared,whole genome methylation,histone H3 acetylation and other acetylation sites,and the expression of epigenetic modification enzymes in them were detected,so that to provide a new experimental basis for understanding growth mechanism and epigenetic differences between adult stem cells and fiber fibroblasts.At the same time,it also could provide a new theoretical basis for the application of adult stem cells.1.Comparison of the growth characteristics of fetal fibroblasts and adult stem cells of Arbas Cashmere goat in vitro.In this study,the expression of pluripotency(NANOG,OCT4,SOX2),proliferation(TERT,PCNA)and apoptosis(P53,BAX)relative genes in three kinds of adult stem cells of Arbas Cashmere goat adipose-derived stem cells(ADSCs),bone marrow stem cells(BMSCs)and muscle-derived satellite cells(MDSCs),was compared with fetal fibroblasts(FFCs)by immunofluorescence,real-time quantitative PCR and Western blot.The results showed that the expression of NANOG,OCT4 and SOX2 were lower in gADSCs,gBMSCs and gMDSCs,and there was no significant difference with gFFCs.The expression of TERT in gBMSCs and gMDSCs was higher than that in gFFCs,whereas its expression in g ADSCs was lower than that in gFFCs.PCNA and BAX were higher expression in gFFCs and gMDSCs,were lower expression in gADSCs and gBMSCs.The relative expression of P53 in gBMSCs was higher,and in gFFCs,gADSCs and gMDSCs were lower.2.Epigenetic modification differences between fetal fibroblast cells and adult stem cells of Arbas Cashmere goatIn order to explore the regulation of epigenetic mechanism of adult stem cells,in this study,Arbas Cashmere goat fetal fibroblasts(FFCs)were used as the control,we analyzed epigenetic patterns of adipose-derived stem cells(gADSCs),bone marrow stromal cells(gBMSCs),and muscle-derived satellite cells(gMDSCs).The results showed that the 5mC content in the genome of gBMSCs was lower than that in the genome of gFFCs,while the 5mC contents in the genome of gADSCs and gMDSCs were higher than that in the genome of gFFCs.The acetylation level of H3K9 was no significant difference between the adult stem cells and gFFCs.Acetylated H3K9,H3K14,H3K18,H4K5 and H4K12 could be detected in gFFCs,gADSCs and gMDSCs,but acetylated H4K5 and H4K12 were almost not detected in gBMSCs.The expression of DNMT1,DNMT3 A and DNMT3 B in g BMSCs and gMDSCs were relatively higher.The expression of TET1 and TET2 in gFFCs,gADSCs,gBMSCs and gMDSCs were relatively lower,and the expression of TET3 was relatively higher but there was no significant differences.The expressions of HDAC1,HDAC6,SIRT1,Tip60 and PCAF in gADSCs,gBMSCs and gMDSCs were all higher than that in gFFCs,which were in accordance with the results of real-time quantitative PCR.But the expression of P300 was not detected.In conclusion,three kinds of adult stem cells and fetal fibroblasts had their own advantages at different levels due to the cells derived from different tissues and age.However,from the overall level,the pluripotency and proliferation capacity of adult stem cells were significantly better than that of fetal fibroblasts.From the perspective of epigenetics,the epigenetic modification of adult stem cells was more active than that of fetal fibroblasts.It was indicated that the active epigenetic modification in adult stem cells was beneficial to its regulation.The results of this study provide new ideas for the application of adult stem cells in the fields of human medicine and animal husbandry.