The Effect Of Hypoxic Preconditioning On The Biological Function Of Bone Marrow-Derived Endothelial Progenitor Cells

Objective:In this study, the in vitro simulated hypoxia micro environment, aims to investigate the effect of hypoxic preconditioning on Bone marrow-derived endothelial progenitor cells(BM-EPCs) morphology, anti-apoptosis ability, in vitro angiogenesis and other biological functions.Methods:1、Under sterile conditions, Bone marorw mononuclear cells(BMMNCs) were obtained from bone marrow of6-8w male Wistar rats (180-220g) by flushing rat’s humerus,tibia,fibula,and femur and were isolated by the density gradient centrifugation. The isolated cells were cultivated in dishes coated with the vitronectin from rat plasma,filled with the specific endothelial cell basal medium-2.2、Randomly aparted into routine training group and hypoxic preconditioning group, cells of the routine training group were cultured under the conventional culture conditions(37℃and5%CO2)for7days. However, cells of the hypoxic preconditioning groups were cultured in conventional culture conditions for4,5and6d, and then under the hypoxic condition (1%O2+5%CO2+94%N2) for another72hours,48hours and24hours, respectively.3、The observation of cell morphology, cell surface marker CD34+/CD133+, Dil labeled acetylated low density lipoprotein and FITC labeled vicia ervilia lectin-1staining, three methods to identify the routine training groups cultured to seventh days of BM-EPCs cells.4、After seven days, cells in all of groups were collected for the following study. Under a microscope, morphological changes of all groups cells is recorded.The tube formation of BM-EPCs was tested by Matrigel assay. Annexin V/PI antiapoptotic assay were used to detect the apoptotic rate of BM-EPCs. Comparison of changes in cell biology function all groups, make clear the hypoxic condition on the impact of BM-EPCs.Results:1、All of groups were isolated and cultured successfully in vitro to the7days. BM-EPCs phagocytosis of Dil-labeled acetylated low density lipoprotein and FITC-labeled Ulex lectin-1double staining cells positive rate (90%±4%), cell surface markers CD34+/CD133+double-positive rate (86%±2%).2、The early apoptotic rate of BM-EPCs was increased obviously with the hypoxic time extended. There was no significant difference in the early apoptotic rate of BM-EPCs between routine training group (0.89±0.20)%and hypoxic24h group (1.33±0.07)%(P>0.05). There were significant increase in the early apoptotic rate of BM-EPCs in hypoxic48h group (3.25±0.12)%and hypoxic72h group (7.48±1.53)%(P<0.05).3、With prolonged hypoxia treatment time, the changes of BM-EPCs in vitro the tube formation abilities is significantly. Compare with routine training group,the tube formation ability was significantly increased in hypoxic24h group (P<0.01), but the tube formation ability was significantly decreased hypoxic48h group and hypoxic72h group (P<0.01). Compare with hypoxic24h group, the tube formation ability was significantly decreased hypoxic48h group and hypoxic72h group (P<0.05).Conclusion:After hypoxic preconditioning for24hours, the apoptotic rate was not obvious in BM-EPCs, but the tube formation ability was markedly increased.