百草枯对体外培养小鼠胚胎神经干细胞增殖的影响
Effects of Paraquat on the Proliferation Viability of Mouse Embryo Neural Stem Cells in Vitro
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摘要:
目的 观察百草枯(paraquat,PQ)对体外培养小鼠胚胎神经干细胞增殖活力的影响。 方法 取10.5 d小鼠胚胎进行体外神经干细胞原代培养,对培养细胞进行形态学观察、特异性蛋白质免疫细胞化学染色鉴定;用终浓度为0.00、0.10、1.00、10.00、100.00、1 000.00μmol/L的PQ处理神经干细胞24 h后,采用阿尔玛蓝细胞增殖与细胞毒检测法(Alamar Blue法)测定细胞相对存活率,分析不同浓度PQ对神经干细胞增殖的影响。用终浓度为0.00、0.10、1.00、10.00、50.00、100.00μmol/L的PQ处理神经干细胞24 h后,用活性氧荧光定量法(DCFH-DA法)检测细胞内活性氧(ROS)水平。 结果 免疫细胞化学检测结果显示,培养的细胞可以表达神经干细胞(NSCs)特异性蛋白;经诱导分化后的细胞可以表达神经细胞特异性蛋白;Alamar Blue法检测结果表明,与对照组相比,当PQ ≥ 100.00 μmol/L时,细胞存活率明显降低;并伴随染毒浓度的升高而进一步降低(P < 0.05)。ROS检测结果表明,与对照组相比,当PQ ≥ 10.00 μmol/L时,细胞内ROS水平明显提高。 结论 培养的神经细胞具备神经干细胞的基本特征;PQ可引起细胞内ROS水平的升高,并抑制神经干细胞的增殖,且存在剂量依赖性。 Abstract:Objective To isolate, cultivate and identify the neural stem cells (NSCs)of mouse embryo in vitro and study the effect of paraqua(t PQ)on their proliferation viability. Methods Cerebral cortexes were isolated from embryonic (E10.5 d) mice. The cells were identified as neural stem cells through immunocytochemistry assey with antibodies to neural stem cells (NSCs) specific protein and morphological observation. After treatment with 0.00, 0.10, 1.00, 10.00, 100.00, 1 000.00 μmol/L PQ for 24 h, the viability of the NSCs was measured by Alamar Blue assay. After treatment with 0.00, 0.10, 1.00, 10.00, 50.00, 100.00μmol/L PQ for 24 h, the reactive oxidative spieces (ROS)production of the NSCs was measured by DCFH-DA assay. Results The result of immunocytochemistry assey showed that nestin which is the specific protein of NSCs was expressed in the majority of the cells. Alamar Blue assey reveals that the cell viability was reduced significantly in the groups of PQ ≥ 100.00 μmol/L compared with the control group (P < 0.05). With the increase concentration of PQ, the cell viability decreased accordingly (P < 0.05). DCFH-DA result showed that the ROS production increased significantly in the groups received PQ ≥ 10.00 μmol/L compared with the control group (P < 0.05). Concentration with the increase of PQ, the ROS production increased accordingly (P < 0.05). Conclusion Primary culture of NSCs in vitro was established successfully. PQ contributed a dose-dependent relationship on suppression of the proliferation and raise of ROS level of NSCs.
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