Effects Of Hypoxia On Proliferation And Differentiation Of NSCs And The Mechanisms

Neural stem cells(NSCs),wich exist in the developing and adult mammalian brain,are self-renewing and can differentiate into neurons,astrocytes or oligodendrocytes.So they have wide prospects of application in treatment of intractable diseases and damaged mammalian CNS,which has been hot point of research in central nervous field. Molecular oxygen (O₂) is vital to nearly all forms of life on earth and is also an important physiological regulator for the cell function.Currently the researches are focused on the hypoxia injury and the hypoxia adaptation, with little effort at the effect of hypoxia on the proliferation and differentiation of stem cells such as NSCs.Hypoxia inducible factor-1 (HIF-1) is the central regulating factor in cellular response to hypoxia.Evidence is mounting that HIF-1 and its downstream targets play pivotal roles in the proliferation of cancer cells.But there is no report about whether HIF-1 participates the effects of hypoxia on NSC proliferation and differentiation.Here,we compared the effects of different oxygen concentrations on NSC proliferation and differentiation and investigated the role of HIF-1 in NSC proliferation and differentiation induced by hypoxia.1. Isolation,culture and identification of NSCs and detection of O₂ concentrationNSCs dissected from wistar rat embryonic day 13 (E13) mesencephalon were mechanically dissociated.Cells were plated on plastic 35mm petri dishes precoated with poly-L-Lycine.After fixation with 4% paraformaldehyde at 4℃ for 2h,cells were indentificated by immunohistochemistry with a mouse monoclonal antibody against Nestin.The results show that almost all of the cells are Nestin positive.So we can using them to perform the following experiments.NSCs were placed in different O₂ concentration environments(20% O₂, 10% O₂,3%O₂, 0% O₂).After culture of 3 days,the PO2 and lactate concentration in the cell culture medium were detected by Blood-gas Analyzer and Automatic Biochemical Analyzer.In tum,PO₂ are 153±7,105±5,80±10 and 65±15 mmHg and lactate concentrations are 0.875±0.251,1.425±0.112,1.825±0.220 and 2.096±0.233 mmol/L.From above results,when cells were cultured under 0% O₂ condition,the lactate concentration are too high to suit cell growth.So we adopted 10% O₂,3% O₂ to investigate the effects of hypoxia on NSC proliferation and differentiation.2. Effects of hypoxia on NSC proliferation and the mechanism...