载神经生长因子纳米粒诱导神经干细胞分化为神经元及PI3K/Akt通路的影响

摘要

背景：课题组前期研究证实NGF-PEG-PLGA-NPs在体外有良好的缓释效能及生物活性，可以诱导PC12细胞向神经元样细胞分化。　　目的：探讨NGF-PEG-PLGA-NPs诱导胎鼠大脑隔区来源神经干细胞分化为神经元的可行性及其对PI3K/Akt信号通路的影响。　　方法：采用优化处方，复乳化溶剂扩散法制备 NGF-PEG-PLGA-NPs。设对照组、NGF 组、NGF-PEG-PLGA-NPs组、LY294002组、LY294002+NGF组、LY294002+NGF-PEG-PLGA-NPs组，诱导神经干细胞分化为神经元，细胞免疫荧光染色对神经元进行鉴定，Werstern-blotting检测PI3K/Akt信号通路Akt磷酸化水平。　　结果与结论：对照组、NGF 组、NGF-PEG-PLGA-NPs 组、LY294002组、LY294002+NGF 组、LY294002+NGF-PEG-PLGA-NPs组β-Tubulin Ⅲ阳性神经元分化率分别为(22.80±2.58)%，(35.80±3.98)%，(35.40±5.77)%，(26.60±3.87)%，(21.20±2.59)%，(25.80±7.22)%。NGF 组与 NGF-PEG-PLGA-NPs 组神经元分化率差异无显著性意义(P>0.05)，但两组神经元分化率均高于其他各组(P<0.05)。Western blotting检测结果显示：NGF组及NGF-PEG-PLGA-NPs组Akt磷酸化水平差异无显著性意义(P >0.05)，但均高于其他各组(P<0.05)；LY294002+NGF组及LY294002+NGF-PEG-PLGA-NPs组Akt磷酸化水平与对照组比较差异无显著性意义(P>0.05)，但均高于LY294002组(P<0.05)。结果表明NGF-PEG-PLGA-NPs可促进神经干细胞分化为神经元，其可能机制与促进PI3K/Akt信号通路Akt的磷酸化有关。%BACKGROUND:Our previous studies confirmed that NGF-PEG-PLGA-NPs has good sustained release effect and biological activity in vitro, and can induce the differentiation of PC12 cel s into neuron-like cel s. OBJECTIVE:To investigate the feasibility of neuronal differentiation of neural stem cel s from septal area of fetal brain induced by NGF-PEG-PLGA-NPs and its influence on PI3K/Akt signaling pathway. METHODS:According to optimization prescription, NGF-PEG-PLGA-NPs were prepared by multiple emulsion solvent diffusion method. Neural stem cel s were induced to neuronal differentiation in six groups, including control group, NGF group, NGF-PEG-PLGA-NPs group, LY294002 group, LY294002+NGF group and LY294002+NGF-PEG-PLGA-NPs group. Neurons were identified by immunofluorescence, while phosphorylation levels of Akt in PI3K/Akt signaling pathway were detected by western blotting. RESULTS AND CONCLUSION:The proportions ofβ-Tubulin III-positive neurons in control group, NGF group, NGF-PEG-PLGA-NPs group, LY294002 group, LY294002+NGF group and LY294002+NGF-PEG-PLGA-NPs group were (22.80±2.58)%, (35.80±3.98)%, (35.40±5.77)%, (26.60±3.87)%, (21.20±2.59)%and (25.80±7.22)%, respectively. There were no statistical differences in neuronal differentiation between NGF group and NGF-PEG-PLGA-NPs group (P>0.05), but the ratios of neural differentiation in the two groups were both higher than that in the other four groups (P<0.05). Western blotting results revealed that there were no statistical differences in Akt phosphorylation levels between NGF group and NGF-PEG-PLGA-NPs group (P>0.05), but the phosphorylation levels of Akt were both higher than other four groups (P<0.05). There were also no significant differences between LY294002+NGF and LY294002+NGF-PEG-PLGA-NPs groups and control group (P>0.05), but the phosphorylation levels of Akt were higher than LY294002 group (P<0.05). Results suggest that NGF-PEG-PLGA-NPs promoted neural differentiation of neural stem cel s. The role might be related to increasing phosphorylation levels of Akt in PI3K/Akt signaling pathway.