Effects Of HIF-1α On Hypoxia Tolerance Of Human Amniotic Mesenchymal Stem Cells After Hypoxia Treatment

ObjectiveThe HIF-1α gene was transiently transfected by Co Cl2-induced hypoxia of human amniotic mesenchymal stem cells(h AMSCs).The survival and apoptosis of h AMSCs after hypoxic treatment were observed,and the expression of HIF-1α,VEGF and apoptosis proteins Bcl-2,Bax and C-Caspase-3 were detected to investigate the effect of HIF-1α gene on h AMSCs tolerance to hypoxia.MethodsIsolate and cultivate primary h AMSCs,Oct-4 and NANOG were detected by cell immunofluorescence assay in h AMSCs;Cocl2 induced h AMSCs hypoxia;The cell survival rate of each group was determined by CCK-8 after anoxic treatment at different time points(12,24,48h);Cell apoptosis rate was detected by flow cytometry in 24 h after hypoxia;HIF-1α,VEGF,Bcl-2,Bax and C-Caspase-3 protein expression were detected by Western blot.Results200umol/l and 24 h were the ideal hypoxia environment for h AMSCs.The result of CCK-8 showed that the cell survival rate of HIF-1α interference group was significantly lower than that of untreated group and no-load interference control group at each time point after hypoxia;while compared with the untreated h AMSCs group and the no-load overexpression control group,the cell survival rate of HIF-1α overexpression group was significantly increased(P <0.05);the cell survival rate in HIF-1α overexpression group was significantly higher at 24 h than that at 12 h and 48 h(P < 0.05).The result of flow cytometry showed that the apoptosis rate of HIF-1α interference group was significantly higher than that of untreated group and no-load interference h AMSCs(P < 0.05),compared with the untreated control group of h AMSCs,the apoptosis rate of hif-1α overexpression group was significantly reduced.(P < 0.05).Western blot results showed that the expression of HIF-1α,VEGF and Bcl-2 protein in HIF-1αinterference group was significantly decreased,while the expression of Bax and C-Caspase-3 protein was significantly increased compared with untreated and empty control h AMSCs group(P < 0.05);On the contrary,Compared with the h AMSCs untreated group and the no-load interference control group,the expression of HIF-1,VEGF and Bcl-2 protein in HIF-1αoverexpressing group increased significantly,and the expression of Bax and C-Caspase-3 protein decreased.ConclusionsHIF-1α gene overexpression improved h AMSCs survival rate and anti apoptosis rate,and improve h AMSCs tolerance to hypoxia.Its possible mechanism is related to up regulation of VEGF and related apoptosis gene Bcl-2 expression and down regulation of Bax and C-Caspase-3 expression.