【摘要】 【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。更多还原

【Abstract】 【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.更多还原