REGULATING EFFECTS OF THE PI3-K/Akt SIGNALING PATHWAY ON THE DIFFERENTIATION OF MARROWDERIVED CARDIAC STEM CELLS

Objective To investigate the regulating effects of PI3-K/Akt signaling pathway on the differentiation of the marrow-derived cardiac stem cells(MCSCs) induced with BMP-2 toward cardiomyocytes and to determine the possible mechanisms in signal transduction on differentiation of MCSCs toward cardiomyocytes.Methods MCSCs were selected from the bone marrow of SD rats with single-cell cloning culture.The differentiation of MCSCs toward cardiomyocytes was induced with 10 μg/L BMP-2,and changes in pAkt levels in MCSCs were examined with Western blotting and immunocytochemistry using several time points after induced with the BMP-2.Effect of PI3-K was detected with the PI3-K inhibitor Ly294002.Expression of Nkx2.5、GATA-4 and cTnT、Cx-43 mRNA of MCSCs before and after induction were determined with RT-PCR.After induction with BMP-2,the morphological alterations to the cells were examined under a phase contrast microscope.Results MCSCs express c-kit and the early cardiac transcription factors Nkx2.5.Before induction with BMP-2,the p-Akt level in MCSCs was low.After induction with BMP-2,the p-Akt level in MCSCs was increased and was to reach a peak at 20 to 30 min and subsequently decreased at 1 hour.After treatment with the PI3K inhibitor Ly294002, p-Akt level of the cells was decreased significantly.RT-PCR detection showed that expression of Nkx2.5 and GATA-4 were weak before induction;however expression of Nkx2.5 and GATA-4 was increased at two weeks and conspicuous at four weeks after induction.Expression of cTnT and Cx-43 mRNA was not observed before induction,and was obvious at two weeks and even stronger at four weeks after induction.After treatment with Ly294002,the expression of Nkx2.5,GATA-4,cTnT and Cx-43 mRNA was reduced significantly,although no morphological change to the cells was obvious.Conclusion BMP-2 may induces differentiation of MCSCs toward cardiomyocytes,and the PI3-K/Akt signaling pathway plays an important role in mediating the differentiation of MCSCs induced with BMP-2 toward cardiomyocytes.