丁苯酞对神经干细胞向神经细胞分化的促进作用

Butylphthalide Promotes the Differentiation of Neural Stem Cells into Neural Cells

关键词:  丁苯酞  神经干细胞  神经细胞  PI3K-AKT

Key words:  butylphthalide  neural stem cells  nerve cells  PI3K-AKT

摘要:

目的 研究丁苯酞通过PI3K-AKT信号通路促进神经干细胞向神经细胞分化的作用。方法 分离培养SD大鼠胚胎脑皮质层的神经干细胞，采用分化专用完全培养基培养后，通过光镜下观察和Nestin蛋白免疫荧光法进行鉴定。设置对照组和实验组，其中实验组添加丁苯酞辅助分化，分为丁苯酞低剂量组（1 μmol·L^-1）、中剂量组（5 μmol·L^-1）和高剂量组（10 μmol·L^-1），作用24 h后，CCK-8法检测神经干细胞的细胞活力，碱性磷酸酶染色法检测神经干细胞的分化能力，免疫荧光法检测神经细胞分化程度，RT-qPCR法检测转录因子SOX2、PAX6和NeuN的mRNA表达水平，Western-blot及RT-qPCR检测PI3K-AKT信号通路的表达。结果 丁苯酞干预后神经干细胞活力提高，分化能力提高，碱性磷酸酶试验呈强阳性，转录因子SOX2、PAX6和NeuN的mRNA表达水平增高，PI3K-AKT信号通路蛋白和mRNA表达水平均增高，且呈现剂量依赖性。结论 丁苯酞可以促进神经干细胞向神经细胞分化，其作用机制可能与PI3K-AKT的激活有关。

Abstract:

OBJECTIVE To study the effect of butylphthalide(NBP) on the differentiation of neural stem cells into neural cells through PI3K-AKT signaling pathway. METHODS The neural stem cells were isolated and cultured from the cerebral cortex of SD rats. Cultivation in complete medium for differentiation. The neural stem cells were identified by light microscopy and Nestin immunofluorescence. Set the control group and the experimental group, the experimental group was supplemented with NBP to assist differentiation and divided into low dose group(NBP 1 μmol·L^-1), medium dose group(NBP 5 μmol·L^-1) and high dose group(NBP 10 μmol·L^-1). After 24 h, the viability of neural stem cells was detected by CCK-8, and the differentiation ability of neural stem cells was detected by alkaline phosphatase staining. The results differentiation of neural stem cells detected by MAP2 immunofluorescence, RT-qPCR detected the mRNA expression levels of transcription factors-SOX2, PAX6 and NeuN. The expression of PI3K-AKT signaling pathway was detected by Western-blot and RT-qPCR. RESULTS The activity of neural stem cells was increased and the differentiation ability of the neural stem cells was strengthened. The alkaline phosphatase test was strongly positive. The expression levels of transcription factors SOX2, PAX6 and NeuN mRNA were increased, and the expression levels of PI3K-AKT signaling pathway protein and mRNA were increased, they all showed a dose-dependent. CONCLUSION NBP can promote the differentiation of neural stem cells into neural cells, and its action may be related to the activity of PI3K-AKT.