Study On Effect Of Mesenchymal Stem Cell Exosomes On Epididymal Epithelial Cells Injury Induced By Ionizing Radiation And Its Mechanism

Objective:This study was aimed to investigate the repairing effect of exosomes derived of mesenchymal stem cells on epididymal epithelial cells injury by ionizing radiation and it’s possible mechanism.Methods:First,human umbilical cord mesenchymal stem cells were cultured,and the exosomes of mesenchymal stem cells were separated by ultracentrifugation.The size of exosomes was analyzed by transmission electron microscopy and NTA,and the expression of exosome specific markers was identified by WB and flow cytometry.Secondly,the primary culture method of mouse epididymal epithelial cells was established by isolating mouse epididymal epithelial cells,and identified by the determination of epididymal epithelial cell specific protein GPX-5,epithelial cell specific protein CK-18,epididymal epithelial cell specific protein sialic acid and α-1Lepididymal 4-glycosidase.Epididymal epithelial cells were irradiated with different doses(0,2,4 and 6Gray).The apoptosis and cell activity of epididymal epithelial cells after ionizing radiation were detected by Annexin-V/PI flow assay and CCK-8,and the expression of y-H2AX,a protein related to radiation injury,was detected by WB and immunofluorescence,so as to establish the model of epididymal epithelial cell injury induced by ionizing radiation.Finally,mesenchymal stem cell exosomes(0GY,0GY+EXO,4GY,4GY+EXO)were added to the radiation injury of epididymal epithelial cells,and different concentrations of mesenchymal exosomes(0,6.25,12.5,25,50,100,100,200μg/ml)were added under the same irradiation condition.The apoptosis and activity of epididymal epithelial cells were detected,and the expression levels of y-H2AX and apoptosisrelated proteins Cleave-Caspase3 and Cleave-PARP were detected.The mechanism of epididymal ionizing radiation injury repair by mesenchymal stem cell exosomes was further explored by detecting the activation of AKT,MAPK and mTOR signal pathways,and using miRNALet-7g and inhibitors rich in exosomes.The experimental data were sorted out and statistically analyzed by Graphpad7.0 software.Multiple groups of data were compared by one-way ANOVA(ANOVNA)and Dunnett’s test.T-test was used between the two groups.When P<0.05,it was considered that the difference was statistically significant.Result:The exosomes of human umbilical cord mesenchymal stem cells were isolated successfully.The results of electron microscope and NTA analysis showed the diameter of the exosome was about 30-150nm.The results of WB showed that all exosomes marker proteins such as CD9,CD63,CD81,Flotilinl and HSP70 were expressed.A stable culture system of primary epididymal epithelial cells was established.The cultured epithelial cells adhered to the wall like pebbles and expressed epididymal epithelial cellspecific protein GPX-5 and epithelial cell-specific protein CK-18.ELISA showed that they could secrete epididymal epithelial cell-specific protein sialic acid and α-1,4-glycosidase.After ionizing radiation,with the increase of radiation dose,the activity of epididymal epithelial cells decreased significantly(t=10.47,P<0.001),apoptosis increased significantly(t=5.552,P<0.001),and the expression of γ-H2AX,a protein related to radiation injury,was up-regulated.After the intervention of exosome,the cell activity was increased(t=45.39,P<0.0001),the apoptosis was inhibited,and the proportion of apoptosis was significantly decreased(t=5.776,P<0.001).With the increase of exosome concentration,the cell activity increased gradually,and there was significant difference when the concentration of exosome was higher than that of 25μg/ml(P<0.001).The expression of γ-H2AX,P-AKT,Cleave-Caspase3 and Cleave-PARP was significantly up-regulated after radiation.After the intervention of exosome,the expression of γ-H2AX,P-AKT,Cleave-Caspase3 and Cleave-PARP was significantly down-regulated.There was no significant change in the expression of MAPK and mTOR.And the expression of γ-H2AX,a protein related to radiation injury,was down-regulated after the addition of miRNA Let-7g mimc,while Let-7g miRNA inhibitor reduced the repair effect of exocrine on radiation injury.Conclusion:Ionizing radiation can induce epididymal epithelial cell apoptosis and reduce epididymal epithelial cell viability;mesenchymal stem cell exocrine can inhibit epididymal epithelial cell apoptosis induced by ionizing radiation,increase cell activity and promote the repair of radiation injury;MSC exosomes can promote the repair of epididymal epithelial cells by miRNA Let-7g,which activates the AKT signaling pathway to regulate the expression of γ-H2AX.