Protective Effects Of Different Concentrations Of Edaravone On Cardiac Stem Cells Under Hypoxic Condition

Objective:1.This study was undertaken to establish a brief and mature method for cell isolation from the adult mice hearts and explore the multipotency characteristic of Cardiac Stem Cells?CSCs?.2.Furthermore,the effects of different concentrations of edaravone on CSCs under hypoxic condition were explored.Methods:1.The spiral condenser,constant flow pump and thermostat water bath were connected to form a simple perfusion system.2.The c-kit+CD45-CSCs cells were isolated by MACs using CD45 and c-kit magnetic microbeads.3.The differentiation of the cells were analyzed by immunofluorescence and RT-PCR.4.The CSCs were seeded in the medium containing edaravone of different concentrations?0?100?300?500?700?1000?mol/L?and cultured in 1%O₂?5%CO₂?94%N₂ incubator.The production of ROS was detected by DFCH-DA.Atoposis of the CSCs was analyzed by Annexin V/7AAD staining.Migration of the CSCs was determined by Transwell.RT-PCR was used to measure the gene expression of bFGF.Results:1.The dissection of the myocardium was complete and the cells were well separated.2.The purity of c-kit+cells was 89.3%and CD45+cells was as low as 0.8%.There was a significant increase in the expression of?-SMA,sm-22?,vWF and cTnT mRNA.However,the expression of c-kit mRNA was significantly decreased at day14 when compared with day0?p<0.05?.vWF positive cells,?-SMA positive cells and cTnT positive cells can be observed by Immunofluorescence.3.When treated with 1000?mol/L edavarone,the ROS production of the CSCs was significantly decreased compared with other groups?p<0.05?.Flow cytometric analysis showed that the apoptosis in 1000?mol/L group was most significantly attenuated compared with the 0,100?mol/L groups?p<0.05?.Edavarone can further enhance the CSCs migration under the hypoxia condition.700?mol/L group exerted the most significant effect on the CSC migration compared with the 0?mol/L group?p<0.05?.The expression of bFGF can be enhanced by edavarone,700?mol/L group exerted most significant effect?p<0.05?.Conclusions:The mice myocardium can be completely dissected into single cells through this simple perfusion system,and the result is well repeated.The isolated c-kit+CD45-CSCs cells possess the characteristic of multipotency.Edaravone has positive effects on CSCs in the ROS,apoptosis,migration and bFGF mRNA expression,in a certain range with a dose-dependent manner.