缺氧损伤对新生大鼠海马神经干细胞细胞活力及分化潜能的影响

The Effect of Hypoxia on the Vigor and Differentiated Ability of Neural Stem Cells from New Born SD Rats

【目的】通过建立缺氧损伤模型,观察缺氧损伤对神经干细胞活力及分化潜能的影响。【方法】采用机械吹打、无血清悬浮培养,95%N2、5%CO2比率的缺氧气体体外分离培养新生大鼠海马神经干细胞,建立缺氧模型,免疫细胞化学鉴定神经干细胞及其分化后的细胞类型,MTT、LDH等方法比较缺氧培养后神经干细胞的变化,并观察缺氧培养1、2、4、6、8 h后神经元分化的比率。【结果】新生大鼠海马可分离出的大量细胞Nestin染色阳性,证实为神经干细胞,分离出的神经干细胞可分化为神经元、胶质细胞等神经细胞类型;95%N2、5%CO2比率的缺氧气体培养6 h后神经干细胞MTT值由缺氧前0.368±0.104降至0.268±0.071(P<0.05),LDH漏出量由缺氧前10.369±0.379 unit/(ml.min)增加到13.987±1.240 unit/(ml.min)(P<0.05);神经元的分化比率稍增加,但无显著差异(P>0.05)。【结论】新生大鼠海马内提取的细胞为神经干细胞,神经干细胞缺氧培养6 h后可以建立神经干细胞缺氧损伤模型,短时间缺氧培养对细胞分化的类型无明显改变。

[Objective]To isolate and culture NSCs from hippocampus of new born SD rats,establish the hypoxia model,and investigate the regulation of oxygen deficiency on the proliferation and orientating differentiation of NSCs.[Methods]The brain tissues of SD rat were dissociated mechanically.Cell suspensions were cultivated with serum-free culture medium by suspension culture technique in order to acquire large numbers of self-proliferating cell clones.Immunocytochemical stainings of anti-Nestin,NF-L,GFAP were used to identify the cells.NSCs were exposed to 5% O2 for 1h,2h,4h,6h or 8h.Proliferation of NSCs were observed after hypoxia by MTT and LDH method and the ratio of the differentiated neurons,morphological changes were detected.[Results]NSCs dissociated from SD rat could differentiate into neurons and astrocytes etc.After 6 hours of hypoxia,the OD value was decreased from（0.368±0.104） before anoxia to（0.268±0.071）,the effluxes of LDH were increased from（10.369±0.379） unit/（ml·min） before anoxia to（13.987±1.240） unit/（ml·min）,but there were no significant changes of the ratio of the differentiated neurons.[Conclusion]NSCs can be dissociated from SD rat,and after 6 hours of anoxia, hypoxia model can be established,there are no significant changes of the ratio of the differentiated neurons.