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[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10.5d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、1000.00μmol/L的PQ处理神经干细胞24h后,采用阿尔玛蓝细胞增殖与细胞毒检测法(AlamarBlue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24h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;AlamarBlue法检测结果表明,与对照组相比,当PQ≥100.00μmol/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≥10.00μmol/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。
[Objective] To observe the effect of paraquat (PQ) on the proliferation of mouse embryonic neural stem cells cultured in vitro. [Method] Primary culture of neural stem cells in vitro was performed on 10.5d mouse embryos. Morphological observation and specific protein immunocytochemical staining were performed on the cultured cells. The cells were treated with final concentrations of 0.00,0.10,1.00,10.00,100.00,1000.00μmol / L PQ treatment of neural stem cells 24h later, using Almar’s blue cell proliferation and cytotoxicity assay (AlamarBlue method) relative cell viability was measured, the effects of different concentrations of PQ on the proliferation of neural stem cells. The neural stem cells were treated with PQ at final concentrations of 0.00,0.10,1.00,10.00,50.00 and 100.00μmol / L for 24 hours. The level of reactive oxygen species (ROS) in the cells was measured by reactive oxygen species-specific fluorescent assay (DCFH-DA). [Results] The results of immunocytochemistry showed that the cultured cells could express NSCs specific proteins; the differentiated cells could express neural cell specific proteins; the results of AlamarBlue assay showed that compared with the control group, When PQ≥100.00μmol / L, the cell viability decreased significantly, and further decreased with the increase of exposure concentration (P <0.05). The results of ROS assay showed that compared with the control group, the level of intracellular ROS was significantly increased when PQ≥10.00μmol / L. [Conclusion] The cultured nerve cells possess the basic features of neural stem cells. PQ can induce the increase of intracellular ROS and inhibit the proliferation of neural stem cells in a dose-dependent manner.