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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 the differentiation of rat epidermal stem cells into neurons in vitro. Methods: The rat MicroRNA-9-1 lentiviral vector was constructed and infected with epidermal stem cells of SD rats. The experiment was divided into infected group, uninfected group and negative control group. After induced by β-mercaptoethanol, Stem cells differentiate into nerve cells. The expression of GFP was detected by inverted fluorescence microscope. The expression of MAP-2 was detected by immunocytochemistry. The expression of MAP-2 mRNA was detected by RT-PCR. Results: The positive cloned PCR showed that the constructed MicroRNA-9-1 lentiviral vector was successfully constructed. The expression of GFP reached the peak at 48 h after infection, and the infection efficiency reached (85.6 ± 1.9)% under the inverted microscope. ; β-mercaptoethanol induced most of epidermal stem cells to differentiate into neurons at 7 h, and the induction effect was significantly better than that of non-infected group and negative control group; MAP-2 protein (87.3 ± 0.6)%) and mRNA The level (about 2 times) expression rate was significantly higher than other groups (P <0.05). Conclusion: MicroRNA-9-1 lentiviral vector can efficiently infect rat epidermal stem cells and promote the differentiation of epidermal stem cells into neurons induced by β-mercaptoethanol.