Ngn2促进皮肤干细胞神经分化的电生理学及其机制研究

Electrophysiology of Neurogenin2 Promoting Neural Differentiation of Skin-derived Precursors and Its Mechanisms

目的:探讨Ngn2基因转染皮肤干细胞(SKPs)促进神经分化的电生理学机制及可能的机制。方法:体外培养SD大鼠乳鼠SKPs并纯化、鉴定。构建包装含Ngn2基因并用绿色荧光蛋白(GFP)标记的病毒载体。将SKPs分3组:Ngn2组为包装有Ngn2基因的慢病毒转染的SKps,空病毒组为未包装任何基因的慢病毒转染的SKps,空白对照组为未经慢病毒转染的SKps,各组6皿。诱导液诱导14 d,全细胞膜片钳技术检测各组SKPs的电生理活动;Wetern Blot检测各组SKps钠离子通道相关蛋白Nav1.3及Notch信号通路相关蛋白Hes1和Dll1表达水平。结果:诱导14 d后,Ngn2组SKPs出现电压依赖性钠离子通道电流,而其他2组SKPs均未引出钠离子通道电流;Ngn2组SKPs的Nav1.3蛋白表达水平高于其他2组(P<0.01);Ngn2组SKPs Dll1蛋白的表达水平高于其他2组,Hes1蛋白的表达水平低于其他2组(P<0.01)。结论:Ngn2基因促使SKPs表达电压门控性钠离子通道,其机制可能与Notch信号通路有关。

Objective： To explore the electrophysiology of neurogenin2 （Ngn2） promoting neural differentiation of skin-derived precursors （SKPs） and its mechanisms. Methods： SKPs were isolated from SD rat skin tis- sue and purified and identified. Lentvirus contained Ngn2 gene were enclosed and used as the vehicle for gene transduction. SKPs were divided into three groups （n=6 respectively）： SKps in Ngn2 group were transfected with Ngn2 gene via GFP-lentivirus; SKps in blank virus group were transfected with only GFP-lentivirus; SKps in control group were cultured without transfection with lentivirus. After 14-days incubation in neuronal induction medium, electrophysiological analyses were performed. The expression level of Navl.3, related to Na＋ channel and the expression levels of Hesl and Dill, ligands and effectors of Notch signaling pathway, were detected by Western Blot analysis. Results： The patch-clamp recordings of SKPs in the Ngn2 group A indicated that Ngn2-SKps expressed voltage-gated Na＋ channel, which could be blocked by 0.5μm TTX. However, SKPs in the other two groups did not show any inward currents. Western Blot analysis demonstrated that the expression levels of Navl.3 and Dill in the Ngn2 group were higher than those in the other two groups （P〈0.01）. However, the expression level of Hesl in the Ngn2 group was significantly lower than those in the other two groups （P〈 0.01）. Conclusion： Ngn2 promotes SKPs to acquire voltage-gated Na＋ channel, usually expressed in neurons, which may be mediated by Notch signaling pathway Key words