The Study Of Effect Of Hypoxia Preconditioned Aged Human Bone Marrow Mesenchymal Stem Cells In Vitro

Objective: Hypoxia preconditioned of bone marrow mesenchymalstem cells before transplantation could promote cell survive in the targettissue. Human bone marrow mesenchymal stem cells, which support repairwhen administered to cerebral ischemia, in large part through secretedfactors that enhances angiogenesis and neurogenesis, yet undefinedmechanisms. The main purpose of this paper is to investigate the effect ofhypoxia preconditioned aged human bone marrow mesenchymal stem cellsin vitro, provide the basis for the treatment of cerebral ischemia.Methods: Aged human (50～70years) bone marrow mesenchymalstem cells were cultured by whole bone marrow adherence method in vitro.The inverted microscope was used to observe morphology and proliferation(3～4generations). Expression of CD90、CD105、CD73and NGB inhuman bone marrow mesenchymal stem cells were shown by flowcytometry. Hypoxia culture was achieved with a humidified atmosphereinside a trigas incubator containing5%CO2,1%O2, and94%N2. Cells atpassage3were incubated in a flask with a density of1×106/ml, cells were incubated at37℃and saturated humidity in an incubator containing1%O2,5%CO2,92%N2for48hours, and those incubated under normal oxygenas controls. The HIF-1α, VEGF, CXCR4, TLR4and NF-κB proteinexpression were determined by Western blot assay. The concentration ofVEGF was measured by ELISA assay in the cell culture supernate. Theki67expression positive cells were detected by immunofluorescence.Results: All of the cells had a fibroblast-like shuttle shapemorphology cultured in vitro and reached80%confluence at10～12days,with the whirlpool-shape. Cultured cells highly expressed CD90, CD105and CD73, but negative for NGB. Hypoxia preconditioned advancesHIF-1α, VEGF, CXCR4, TLR4and NF-κB expression in bone marrowmesenchymal stem cells (P<0.05). Compared with the control group, theVEGF concentration of the hypoxia preconditioned cell culture supernatewas increased significantly (P<0.05). Ki67staining was not significantlyenhanced by hypoxia preconditioned.Conclusion: Hypoxia preconditioned can upregulate the expression ofHIF-1α, VEGF, CXCR4, TLR4and NF-κB, and increase the secretion ofVEGF in aged human BMSCs. Which suggest that hypoxia preconditionedenhances the therapeutic potential of aged human mesenchymal stem cellsin ischemia brain injury.