Effects Of5-azaC On Stem Cell Antigen-1Expression In Murine Endothelial Progenitor Cells

Objective:To observe the stem cell antigen-1(Sca-1) protein expression on murine endothelial progenitor cells(EPCs).To investigate whether CSE and5-azaC can affect EPCs'proliferation capacity and Sca-1protein expression in vitro, and analysis the relationship between EPCs' proliferation capacity and Sca-1protein expression.To investigate whether CSE and5-azaC affect Sca-1protein expression through Sca-1DNA methylation mechanism in its promoter region.Methods:EPCs were isolated from murine bone marrow by density gradient centrifugation and cultured in endothelial growth medium-2(EGM-2). Cells were identified as EPCs:cells with "slabstone"-like appearance, expression of CD34,CD133,Flk-1, taking up Dil-acLDL and combining with FITC-UEA-I. EPCs' proliferation capacity was detected by MTT method. Sca-1protein expression was detected by western-blot.Sca-1DNA methylation sequence in promoter region was detected by BSP method.Results:(1)The amphophilic cells' positive rate after7days' culture was (94.67±4.16)%.The positive rate of the7th day adherent cells,which expressed FITC-CD34. PE-CD133and APC-Flk-1,was (95.07±1.73)%,suggesting these cells as EPCs. The positive rate of the7th day adherent cells,which expressed FITC-CD3,PE-CD133,APC-Flk-1and PerCP-Sca-1,was (94.00±1.67)%.The EPCs expressing PerCP-Sca-1were (98.87±0.24)%.(2)EPCs'proliferation capacity was detected by MTT method.The OD value represented the activity of mitochondria,that is the number of living cells.When CSE interfered EPCs for3hours,compared with the control group,the OD value of1%CSE group and2.5%CSE group increased significantly(P<0.05).With the increasing of CSE concentration, the OD value of5%CSE group and10%CSE group decreased significantly(P<0.05).When CSE interfered EPCs for6hours, compared with the control group, the OD value of1%CSE group increased significantly(P<0.05). With the increasing of CSE concentration, the OD value of2.5%CSE group,5%CSE group and10%CSE group decreased significantly(P<0.05). When CSE interfered EPCs for24hours, compared with the control group, the OD value in all the CSE groups decreased(P<0.05).(3) Compared with the control group, the OD value of1%CSE group,1%CSE+2umol/L5-azaC group,1%CSE+5umol/L5-azaCgroup decreased(P<0.05). Compared with the CSE group, the OD value of 1%CSE+2umol/L5-azaC group,1%CSE+5umol/L5-azaC group increased significantly(P<0.05).There was no statistical difference of the OD value in1%CSE+2umol/L5-azaC group and1%CSE+5umol/L5-azaC group (P>0.05)。(4) Compared with the control group,Sca-1protein expression decreased significantly in CSE group (P<0.05). There was no statistical difference of Sca-1protein expression in AZA group and CSE+AZA group (P>0.05). Compared with the CSE group, Sca-1protein expression increased significantly in AZA group and CSE+AZA group(P<0.05). There was no statistical difference of Sca-1protein expression in AZA group and CSE+AZA group (P>0.05)(5) There was no statistical difference of Sca-1average methylation rate in promoter region among the control group,CSE group, AZA group and CSE+AZA group (P>0.05)Conclusions:Sca-1protein expressed highly on the surface of murine EPCs.With CSE intervention in vitro,EPCs' proliferation capacity increased in the early stage,and decreased in the late stage.5-azaC could improve EPCs'proliferation capacity which was inhibited by CSE in vitro. With CSE and5-azaC intervention in vitro, Sca-1protein expression change had the same trend.with EPCs'proliferation capacity change.5-azaC could regulate Sca-1protein expression.However,whether its demethylation cite located on Sca-1DNA promoter region,or Sca-1histone or Sca-1upstream regulating factor,need further investigation.