糖氧剥夺对神经干细胞增殖、分化及凋亡的影响

摘要

BACKGROUND:Hypoxia is one of the main pathological factors of nervous system diseases, which plays numerous regulatory roles in survival, migration, differentiation, apoptosis of endogenous neural stem cels and exogenous neural stem cels after transplantation. OBJECTIVE:To systematicaly observe the effects of oxygen-glucose deprivation on the proliferation, differentiation and apoptosis of neural stem cels. METHODS:Neural stem cels were isolated from the olfactory bulb of newborn Kunming mice, to establish oxygen-glucose deprivation models. Immunofluorescence technique was used to determine the differentiation ability of neural stem cels subject to oxygen-glucose deprivation, MTT method was used to measure the proliferative ability of neural stem cels under normoxic conditions after oxygen-glucose deprivation for 24 hours and 48 hours, and Hochest 33258 staining was performed to detect the apoptosis of neural stem cels after oxygen-glucose deprivation for 24 hours and 48 hours. RESULTS AND CONCLUSION:Passage 4 neural stem cels were positive for CD133. Compared with the normoxic group, MTT showed that the proliferation of neural stem cels was significantly lower in the oxygen-glucose deprivation group (P < 0.05), moreover, the proliferative ability of neural stem cels was lower in the 48-hour oxygen-glucose deprivation group than the 24-hour oxygen-glucose deprivation group (P < 0.05). The n number of GFAP and β-Tubulin III positive cels was significantly lower in the 48-hour oxygen-glucose deprivation group than the normoxic group (P < 0.05). Apoptotic rate was significantly increased after oxygen glucose deprivation for 48 hours as compared with oxygen-glucose deprivation for 24 hours (P < 0.01) and normoxic group (P < 0.01). These results indicate that oxygen-glucose deprivation have adverse effects on neural stem cel proliferation, differentiation and apoptosis, and its impact depends on the concentration and time of hypoxia as wel as the ability of tolerance to hypoxia.%背景：缺氧作为神经系统疾病发展过程中诸多病理因素中最常见的因素之一，对于内源性神经干细胞以及移植后外源性神经干细胞的存活、迁移、分化、凋亡发挥着诸多调控作用。目的：系统观察糖氧剥夺对神经干细胞增殖、分化、凋亡的影响。方法：从新生鼠嗅球分离培养出神经干细胞，建立糖氧剥夺模型，免疫荧光技术检测糖氧剥夺后神经干细胞分化能力，MTT法检测糖氧剥夺24，48 h后恢复正常条件培养对神经干细胞增殖能力的影响，Hochest33258荧光染色检测糖氧剥夺24，48 h后神经干细胞凋亡状况。结果与结论：第4代神经干细胞CD133免疫荧光鉴定结果呈阳性表达，MTT细胞增殖能力检测结果示：糖氧剥夺组增殖能力明显低于常氧组(P <0.05)，糖氧剥夺48 h组增殖能力低于糖氧剥夺24 h组(P <0.05)。GFAP、β-TubulinⅢ细胞免疫荧光染色结果示糖氧剥夺48 h后神经干细胞分化为星形胶质细胞、神经元总数明显低于常氧组(P<0.05)。Hochest33258染色凋亡率检测结果示糖氧剥夺组细胞凋亡率明显高于常氧组(P <0.01)，糖氧剥夺48 h组神经干细胞凋亡率显著高于糖氧剥夺24 h组(P <0.01)。上述结果表明糖氧剥夺对神经干细胞增殖、分化、凋亡造成不利影响，影响程度取决于缺氧浓度、缺氧时间及自身对于缺氧的耐受性。