Age-related Changes Of Marrow-derived Cardiac Stem Cells And Regulatory Effects Of IGF-1 On Senescence Of Stem Cells

Objective To investigate the changes in ultrastructure and senescence of the marrow-derived cardiac stem cells(MCSCs) from rats at different ages,clarify the impacts of age on proliferation,survival and differentiation of the cells and explore the regulatory effects of IGF-1 on senescence of MCSCs.Methods MMSCs were isolated from bone marrow of young,adult and aged male SD rats.MCSCs were selected from MMSCs with single-cell cloning culture,c-kit immunoflourence staining and RT-PCR analysis.Ultrastructural changes of the cells were viewed with a transmission electron microscope.The senescence-associated changes were examined with SA-P-galactosidase staining and ROS staining.Distribution of cell cycle of the cells was evaluated with flow cytometric analysis.The cells were treated with serum deprivation and hypoxia for 12 hours.The survived and apoptotic cells were determined by Annexin V/PI flow cytometric analysis and Hochest 33342 staining.Differentiation of MCSCs toward cardiomyocytes was induced with BMP-2.Expression of cardiac transcription factors and cardiac specific genes of the cells after induction were examined with RT-PCR.cTnT expression of the cells was examined with immunocytochemistry.In another experiment,the MCSCs of the aged group were treated with IGF-1.The senescence-associated changes were examined with SA-P-galactosidase staining and ROS staining.Distribution of cell cycle was evaluated with flow cytometric analysis.The cells were treated with serum deprivation and hypoxia for 12 hours and the apoptotic cells were determined by Hochest 33342 staining.After induction with BMP-2,expression of cardiac transcription factors and cardiac specific genes in IGF-1 treated group and untreated group were determined with RT-PCR.cTnT expression of the cells was examined with immunocytochemistry.Expression of p53,p21 and bax mRNA and expression of VEGF,HGF and bFGF mRNA were examined with RT-PCR.Results The nucleus/plasma ratio of MCSCs from the aged rats decreased and there were some myelin bodies in the cells of the aged group.With increasing of age,the cells in S+G2/M phase reduced,andβ-galactosidase-positive cells and ROS-positive cells increased.After being treated with serum deprivation and hypoxia for 12 hours, survival rate of the cells from the aged rats was lower than that of the cells from young rats.At four week after induction with BMP-2,expression of Nkx2.5, GATA-4,cTnT mRNA and Cx-43 mRNA of the cells in the young group increased significantly.In adult and aged group,the level of expression of the cardiac transcription factors and cardiac specific genes was lower than that of the cells in young group.In immunocytochemical staining,cTnT expression of the cells in the young group was obvious after induction with BMP-2.Being compared with young group,cTnT expression of cells in the aged group was weak after induction.After treatment with IGF-1,the cells in S+G2/M phase increased in the aged group,β-galactosidase-positive cells and ROS-positive cells reduced.After being treated with serum deprivation and hypoxia for 12 hours,rates of the apoptotic cells in IGF-1 treated group was lower than that of the cells in the untreated group.Being compared with the IGF-1 untreated group,expression of the cardiac transcription factors and cardiac specific genes in IGF-1 treated group was strong after induction with BMP-2.Expression of VEGF,HGF and bFGF mRNA in IGF-1 treated group was higher than that of the cells in untreated group and expression of p53,p21 and bax mRNA in IGF-1 treated group decreased significantly.Conclusion With increasing of age,MCSCs become senescent,the abilities of proliferation,survival and differentiation of the cells decrease.IGF-1 may attenuate senescence of the aged cells and improve their potential of proliferation,survival and differentiation.