miR-146b促进诱导多能干细胞向神经元样细胞的分化

摘要

背景:如何促进诱导多能干细胞向神经元样细胞分化对于神经损伤修复和神经再生具有重要意义.目的:探讨 miR-146b 在诱导多能干细胞分化过程中的表达水平,及其对诱导多能干细胞向神经元样细胞分化的影响.方法:小鼠诱导多能干细胞向神经元样细胞分化7 d后,利用实时荧光定量PCR方法检测多能干细胞特异蛋白和miR-146b的表达;通过过表达miR-146b慢病毒感染诱导多能干细胞,再诱导其向神经元样细胞分化,利用细胞免疫荧光染色法和荧光定量PCR检测干细胞特异蛋白和神经外胚层特异蛋白的表达,以及神经细胞标记分子Tuj-1的表达.结果与结论:①与未分化小鼠诱导多能干细胞相比,向神经元样细胞分化7 d后,干细胞特异表达蛋白Nanog、Oct4和Sox2表达显著下调,而miR-146b表达显著升高;②诱导多能干细胞过表达miR-146b后,Oct4表达显著下降,Nanog和Sox2表达未发生显著变化;同时,在神经分化过程中,过表达miR-146b诱导多能干细胞Nanog、Oct4和Sox2的表达显著下调,神经外胚层特异蛋白Nestin、Sox1和Pax6的表达显著上调;③神经分化培养18 d后,miR-146b显著增加了Tuj-1阳性细胞数;④结果表明,miR-146b可以促进诱导多能干细胞向神经元样细胞方向分化.%BACKGROUND: To facilitate the neuronal differentiation of induced pluripotent stem cells (iPSCs) is of great benefit for the repair of nerve injury and nerve regeneration. OBJECTIVE: To investigate the expression of miR-146b in iPSCs differentiation, and its effects on the neural differentiation of iPSCs. METHODS: After 7-day neural induction of mouse iPSCs, the expressions of miR-146b and pluripotent stem cell markers were detected by real-time PCR. Then, we generated miR-146b overexpressing iPSCs followed by the neural induction. The expression of stem cell and neuroectoderm markers were examined by cell immunofluorescence and real-time PCR respectively. Meanwhile, the expression of Tuj-1, a nerve cell marker, was also detected. RESULTS AND CONCLUSION: Compared with the normal iPSCs, the expressions of pluripotent stem cell markers Nanog, Oct4 and Sox2 were significantly down-regulated in iPSCs undergoing 7-day neural induction, while the level of miR-146b was increased obviously. In miR-146b-overexpressing iPSCs, we found that the expression of Oct4 was substantially decreased, while there were no statistical changes in the expression of Nanog and Sox2. Meanwhile, during the neural differentiation, miR-146b overexpression significantly down-regulated the expression of Nanog, Oct4 and Sox2, and up-regulated the expression of neuroectoderm markers, Nestin, Sox1 and Pax6. After 18 days of neural induction, the miR-146b overexpression significantly increased the number of Tuj-1-positive cells. Taken together, miR-146b plays a vital role in facilitating the differentiation of iPSCs into neuron-like cells.