MicroRNA-9-1促进大鼠表皮干细胞分化为神经细胞

Promoting Effect of MicroRNA-9-1 on Differentiation of Epidermal Stem Cells into Neural Cells in Rats

目的:探讨MicroRNA-9-1在体外诱导大鼠表皮干细胞向神经细胞分化过程中的作用。方法:构建大鼠MicroRNA-9-1慢病毒载体,并感染SD大鼠的表皮干细胞;实验分为感染组、未感染组和阴性对照组;采用β-巯基乙醇诱导感染后的SD大鼠表皮干细胞分化为神经细胞。倒置荧光显微镜下观察表皮干细胞感染后GFP荧光表达的情况;免疫细胞化学染色检测神经微管结合蛋白2(MAP-2)的表达水平;RT-PCR检测MAP-2mRNA的表达水平。结果:阳性克隆PCR检测证明大鼠MicroRNA-9-1慢病毒载体构建成功;感染后48 h,在倒置显微镜下观察到,感染组GFP荧光表达达到峰值,感染效率达到了(85.6±1.9)%;β-巯基乙醇诱导7 h时,感染组大部分表皮干细胞向神经细胞分化,且诱导效果显著好于未感染组和阴性对照组;MAP-2在蛋白((87.3±0.6)%)和mRNA水平(约2倍)的表达率显著高于其他各组(P<0.05)。结论:MicroRNA-9-1慢病毒载体可以高效感染大鼠表皮干细胞,且可以促进表皮干细胞在β-巯基乙醇的诱导下向神经细胞分化。

Objective：To investigate the role of MicroRNA-9-1 in inducing epidermal stem cells（ESCs） differentiation into neurons.Methods：The lentiviral of MicroRNA-9-1 was constructed and transfected into rats epidermal stem cells.The experiment was divided into transfected group,non-transfected group and the negative control group.The β-mercaptoethanol was as an inducer for triggering the ESCs to differentiate into neurons.The GFP fluorescence expression of epidermal stem cells after transfection was observed under inverted fluorescence microscope.The protein and mRNA expression level of microtuble-associated protein 2 （MAP-2） was detected by immunocytochemical method and RT-PCR,respectively.Results：The result of Positive clone PCR confirmed successful construction of MicroRNA-9-1 in rats.Transfection after 48 h,the expressing of GFP fluorescence at peak in transfected group,and transfection efficiency reached （85.6＋1.9）%.Most ESCs differentiated into neurons in transfected group after β-mercaptoethanol induction 7 h,and the effect was significantly better than non-transfected group and the negative control group.The protein （（87.3± 0.6）%） and mRNA （about twice over） expression levels of MAP-2 in transfected group was higher than those in non-transfected group and the negative control group （P〈0.05）.Conclusion：The lentiviral of MicroRNA-9-1 has high transfection efficiency in rats ESCs,and could promoted ESCs differentiate into neurons under β-mercaptoethanol induced.