丁苯酞对神经干细胞向神经细胞分化的促进作用
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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(1μmol·L~(-1))、中剂量组(5μmol·L~(-1))和高剂量组(10μmol·L~(-1)),作用24h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。
OBJECTIVE To study the effect of butylphthalide(NBP) on the differentiation of neural stem cells into neural cells through PI3 K-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 PI3 K-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 PI3 K-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 PI3 K-AKT.
OBJECTIVE To study the effect of butylphthalide(NBP) on the differentiation of neural stem cells into neural cells through PI3 K-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 PI3 K-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 PI3 K-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 PI3 K-AKT.
引文
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[11]FAN Y X,SHANG J Q,CHEN X H,et al.Effects of butyphthalide soft capsules on cognitive function of patients with acute ischemic stroke and its correlation analysis[J].Chin J New Drugs(中国新药杂志),2018,27(8):905-909.
[12]XIE H L,WANG L M,HAN M,et al.Meta-analysis of effect of dl-3-butylphthalide injection on neurological function of patients with acute cerebral infarction[J].Chin J New Drugs Clin Rem(中国新药与临床杂志),2016,35(7):492-498.
[13]LEI H,ZHAO C Y,LIU D M,et al.L-3-n-Butylphthalide attenuatesβ-amyloid-induced toxicity in neuroblastoma SH-SY5Y cells through regulating mitochondrion-mediated apoptosis and MAPK signaling[J].J Asian Nat Prod Res,2014,16(8):854-864.
[14]LIU S S,CAO C Z,LI Z L.Approach to estimation and prediction for normal boiling point(NBP)of alkanes based on a novel molecular distance-edge(MDE)Vector,λ[J].J Chem Inf Comput Sci,1998,38(3):387-394.
[2]杨顺利.丁苯酞注射液治疗急性脑梗死临床疗效分析[J].淮海医药,2017,35(3):307-309.
[3]DULKEN B W,LEEMAN D S,BOUTET S C,et al.Single-cell transcriptomic analysis defines heterogeneity and transcriptional dynamics in the adult neural stem cell lineage[J].Cell Rep,2017,18(3):777-790.
[4]王健,董为伟.急性缺血性脑卒中的神经保护治疗[J].中国现代神经疾病杂志,2003,34(3):133-136.
[5]RABER J,FAN Y,MATSUMORI Y,et a1.Irradiation attenuates nenrogenesis and exacerbates ischemia-induced deficits[J].Ann Neurol,2004,55(3):38l-389.
[6]TAKASAWA K,KITAGAWA K,VAGITA Y,et al.Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats[J].Cereb Blood HMMetab,2002,22(3):299-307.
[7]ARVIDSSON A,COLLIN T,KIRIK D,et a1.Neuronal replacement from endogenous precursors in the adult brain after stroke[J].Nat Med,2002,8(9):963-970.
[8]YU M,LI W,LUO S,et al.Folic acid stimulation of neural stem cell proliferation is associated with altered methylation profile of PI3K/Akt/CREB[J].J Nutr Biochem,2014,25(4):496-502.
[9]HU J G,WANG Y X,WANG H J,et al.PDGF-AA mediates B104CM-induced oligodendrocyte precursor cell differentiation of embryonicneural stem cells through Erk,PI3K,and p38 signaling[J].J Mol Neurosci,2012,46(3):644-653.
[10]KENDALL S E,NAJBAUER J,JOHNSTON H F,et al.Neural stem cell targeting of glioma is dependent on PI3Ksignaling[J].Stem Cells,2008,26(6):1575-1586.
[11]FAN Y X,SHANG J Q,CHEN X H,et al.Effects of butyphthalide soft capsules on cognitive function of patients with acute ischemic stroke and its correlation analysis[J].Chin J New Drugs(中国新药杂志),2018,27(8):905-909.
[12]XIE H L,WANG L M,HAN M,et al.Meta-analysis of effect of dl-3-butylphthalide injection on neurological function of patients with acute cerebral infarction[J].Chin J New Drugs Clin Rem(中国新药与临床杂志),2016,35(7):492-498.
[13]LEI H,ZHAO C Y,LIU D M,et al.L-3-n-Butylphthalide attenuatesβ-amyloid-induced toxicity in neuroblastoma SH-SY5Y cells through regulating mitochondrion-mediated apoptosis and MAPK signaling[J].J Asian Nat Prod Res,2014,16(8):854-864.
[14]LIU S S,CAO C Z,LI Z L.Approach to estimation and prediction for normal boiling point(NBP)of alkanes based on a novel molecular distance-edge(MDE)Vector,λ[J].J Chem Inf Comput Sci,1998,38(3):387-394.