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[目的]观察抑制信号传导和转录激活子3(signal transducer and activator of transcription 3,STAT3)信号通路对神经干细胞(neuronal stem cells,NSCs)的分化及Notch1表达的影响,进一步明确STAT3在调控NSCs分化过程中的作用及与Notch1的协同关系。[方法]从孕14～15 d的SD大鼠胚胎大脑皮质中分离NSCs,体外培养传代。STAT3抑制剂为S31-201。分组:对照组与三个不同浓度的S31-201组(2.5μmol/L、5μmol/L、10μmol/L)。体外培养48 h后,用Western blotting方法观察S31-201对NSCs中p-STAT3蛋白表达的影响;体外培养48 h与72 h后,用细胞免疫荧光化学染色方法观察S31-201对NSCs中微管相关蛋白2(microtubule-associated protein2,MAP-2)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达的影响;体外培养48 h与72 h后,用RT-PCR与Western blotting方法观察S31-201对NSCs中Notch1基因与蛋白表达的影响。[结果]三个浓度的S31-201组均观察到NSCs中p-STAT3表达的下调,以10μmol/L浓度组抑制作用最强(P<0.05)。S31-201在48 h与72h两个时间点均能促进NSCs中MAP-2表达增加并抑制NSCs中GFAP的表达(P<0.05)。S31-201在48与72 h两个时间点均能抑制NSCs中Notch1 mRNA与蛋白的表达(P<0.05)。[结论]S31-201可以有效抑制NSCs中p-STAT3的水平;抑制p-STAT3的表达能促进NSCs向神经元分化并抑制NSCs向星形胶质细胞分化;抑制p-STAT3可下调Noteh1的表达,二者可能协同调控NSCs的分化。
[Objective] To observe the effect of signal transducer and activator of transcription 3 (STAT3) signaling pathway on the differentiation of neural stem cells (NSCs) and the expression of Notch1, and further clarify the role of STAT3 in the regulation of NSCs differentiation Role in the process and synergies with Notch1. [Methods] NSCs were isolated from the cerebral cortex of SD rats embryo 14 to 15 days pregnant and subcultured in vitro. STAT3 inhibitor is S31-201. Grouping: Control group and three different concentrations of S31-201 group (2.5μmol / L, 5μmol / L, 10μmol / L). After cultured in vitro for 48 h, the effect of S31-201 on the expression of p-STAT3 protein in NSCs was observed by Western blotting. After cultured for 48 h and 72 h, the immunofluorescence staining was used to observe the effect of S31-201 on the microtubules (MAP-2) and glial fibrillary acidic protein (GFAP). After 48 h and 72 h culture, the expression of S31 -201 on Notch1 Gene and Protein Expression in NSCs. [Results] The down-regulation of p-STAT3 expression in NSCs was observed in all the three concentrations of S31-201 group. The strongest inhibitory effect was found in 10μmol / L group (P <0.05). S31-201 could increase the expression of MAP-2 and inhibit the expression of GFAP in NSCs at both 48 h and 72 h (P <0.05). S31-201 inhibited the expression of Notch1 mRNA and protein in NSCs at both 48 and 72 h (P <0.05). [Conclusion] S31-201 can effectively inhibit the level of p-STAT3 in NSCs; inhibiting the expression of p-STAT3 can promote the differentiation of NSCs into neurons and inhibit the differentiation of NSCs to astrocytes; inhibiting the expression of Noteh1 by p-STAT3 , Both of which may coordinate the differentiation of NSCs.