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探讨缺氧对体外培养的鼠胚大脑皮质神经干细胞(neural stem cells,NSCs)增殖、分化及凋亡的影响,为NSCs移植治疗缺血缺氧性脑损伤病变提供实验依据。本研究对孕14d(E14)大鼠取鼠胚脑皮质悬浮培养、贴壁诱导分化。取悬浮培养的第三代NSCs分为实验组(10%O2、5%O2)和正常对照组(20%O2),实验组又以缺氧干预的时间不同分为24、72、120h3个组。通过MTT法和BrdU标记法检测缺氧对NSCs增殖的影响,采用caspase-3检测细胞凋亡情况。缺氧培养后再用10%胎牛血清培养基进行诱导分化,用MAP2免疫荧光染色检测缺氧对NSCs向神经元方向分化的影响。结果显示:(1)10%O272h组和5%O272h组均可诱导NSCs增殖,尤以前者最为明显;(2)10%O2120h组和5%O2120h组均可致NSCs凋亡;(3)5%O2组尤以72h组可诱导NSCs向神经元方向分化。本研究结果提示缺氧可影响NSCs的增殖、分化和存活,适度缺氧可诱导离体培养的大鼠脑皮质NSCs增殖,并可向神经元方向分化,而缺氧时间延长可致NSCs凋亡。
To investigate the effects of hypoxia on the proliferation, differentiation and apoptosis of neural stem cells (NSCs) cultured in vitro and to provide experimental evidence for NSCs transplantation in the treatment of hypoxic-ischemic brain damage. In this study, embryonic 14d (E14) rats were cultured in mouse cerebral cortex suspension, adherent differentiation induced. The third generation NSCs in suspension culture were divided into experimental group (10% O2, 5% O2) and normal control group (20% O2). The experimental groups were divided into 24, 72 and 120h groups . The effect of hypoxia on the proliferation of NSCs was detected by MTT assay and BrdU labeling method. The apoptosis of cells was detected by caspase-3 assay. After hypoxia culture, the cells were induced to differentiate by 10% fetal calf serum medium. The effect of hypoxia on the differentiation of NSCs into neurons was detected by immunofluorescence staining with MAP2. The results showed that: (1) The proliferation of NSCs was induced by 10% O272h and 5% O272h groups, especially the former; (2) The apoptosis of NSCs was induced by 10% O2120h and 5% O2120h groups; The% O2 group, especially the 72h group, could induce the NSCs to differentiate into neurons. The results suggest that hypoxia can affect the proliferation, differentiation and survival of NSCs. Moderate hypoxia can induce the proliferation of rat cortical NSCs cultured in vitro and differentiate into neurons. However, hypoxia prolongs the apoptosis of NSCs .