Effects Of Low Oxygen Concentration On Proliferation Of Rat Bone Marrow-derived Mesenchymal Stem Cells In Vitro

Objective:This study is focused on exploring effects of different oxygen concentrations on the proliferation of rat bone marrow-derived mesenchymal stem cells(BMSCs)in vitro,in order to optimize the culture system of MSCs and provide an experimental data base for the further physiological and physiopathological investigations associated with oxygen tension in the stem cells.Methods:The gaseous atmospheres of cell culture were created by using an innovation method of exhausting and gassing in a quantitative manner.The passaged rat BMSCs were exposed to the gaseous conditions of 1%,5%and18%oxygen,respectively,for the indicated length of time.In-situ cell counting,CCK-8 assay,flow cytometry and immunofluorescence staining were employed to evaluate the effects of different oxygen concentrations on the proliferation,cell viability and apoptosis in BMSCs.Furthermore,western blotting was performed to quantify the expressions of hypoxia-inducible factor-la(HIF-1a),cellular tumor protein p53(p53)and proliferating cell nuclear antigen(PCNA),and the phosphorylation of extracellular regulated protein kinase 1/2(ERK1/2)in BMSCs cultured at different concentrations of oxygen.Results:In the comparison with 18%O2,both of 1%and 5%O2 exposures enhanced the proliferation of BMSCs and 1%O2 exposure showed a greater effect,based on the growth curves of these stem cells cultured respectively in the gaseous environments of three oxygen concentrations over 7 days.The stastistical analysis indicated the significant difference of growth curve data between 1%and 18%O2 groups,and so did between 1%and 5%02 groups.After the rat BMSCswere grown under the gaseous conditions of 1%,5%and 18%oxygen for 48h,the cell viability,mitotic index,proliferation index,phosphorylation level of ERK 1/2 protein,expressions of HIF-1a and PCNA proteins increased in both 1%and 5%02 groups,and the increased amplitudes of these measurements in 1%O2 group were higher than those in 5%O2 group.The differences of the said measurements between 1%and 18%02 groups were statistically significant.No stastistically significant differences of apoptosis indexes and p53 expression levels of BMSCs were observed among 1%,5%and 18%02 groups.Conclusion:In the range of 1%to 18%02 in the gaseous atmospheres of cell culture,lower oxygen concentrations have promoting effects on the proliferation of passaged BMSCs deriving from the bone marrow of rats,and 1%02 has a greater effect than does 5%O2.The mechanisms underlying proliferation-promoting action of the low oxygen on the passaged BMSCs are associated with the upregulation of HIF-la and PCNA expressions,and the increase of ERK1/2 phosphorylation.