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目的:建立NTera2/CloneD1细胞向神经元分化的模型,检测神经元限制性沉默因子(NRSF)经分化培养基诱导后表达的变化。方法:收集正常培养的NTera2/CloneD1细胞及经全反式维甲酸(RA)、阿糖胞苷(AraC)、尿苷分阶段诱导共28 d的细胞,显微镜下观察诱导前后细胞的形态学变化;免疫荧光法检测NTera2/CloneD1细胞诱导前后干性标志Nestin、Sox2和成熟神经元特异性标志NF-200、β-tubulinⅢ的表达情况;应用RT-PCR和免疫荧光法对NRSF进行mRNA和蛋白水平的检测。结果:显微镜下观察到正常培养的NTera2/CloneD1细胞呈克隆样生长,经分化培养基诱导后的NTera2/CloneD1细胞表现出典型的神经元样细胞形态。免疫荧光检测表明,未诱导的NTera2/CloneD1细胞表达神经干细胞的标志Sox2、Nestin,不表达成熟神经元特异性蛋白NF-200、β-tubulinⅢ;而经RA等诱导分化的细胞则不表达Sox2、Nestin,表达NF-200、β-tubulinⅢ。RT-PCR和免疫荧光检测显示,NRSF在诱导分化后的NTera2/CloneD1细胞中的表达量显著降低。结论:建立了NTera2/CloneD1细胞向神经元分化的模型,NRSF在诱导后的NTera2/CloneD1细胞中表达量显著下调,提示NTera2/CloneD1细胞在诱导过程中可能通过下调NRSF,使受到NRSF负性调控的神经元特异性蛋白启动表达并上调,进而实现NTera2/CloneD1细胞向神经元的定向分化。
OBJECTIVE: To establish a model of differentiation of NTera2 / CloneD1 cells into neurons and to detect the expression changes of NRSF induced by differentiation medium. Methods: The normal cultured NTera2 / CloneD1 cells and all-trans retinoic acid (RA), cytarabine (AraC) and uridine were induced in different phases for 28 days. The morphological changes of the cells were observed under microscope The expression of Nestin, Sox2 and mature neuron specific markers NF-200 and β-tubulinⅢ in NTera2 / CloneD1 cells before and after induction were detected by immunofluorescence. The mRNA and protein levels of NRSF were detected by RT-PCR and immunofluorescence The test. Results: The normal cultured NTera2 / CloneD1 cells were observed to grow in a clonal manner under the microscope. The NTera2 / CloneD1 cells induced by the differentiation medium showed typical neuron-like cell morphology. Immunofluorescence showed that Sox2 and Nestin, the markers of neural stem cells, were not expressed in NTera2 / CloneD1 cells, but not mature neuron-specific proteins NF-200 and β-tubulinⅢ. Cells differentiated by RA and so on did not express Sox2, Nestin, express NF-200, β-tubulin Ⅲ. RT-PCR and immunofluorescence showed that the expression of NRSF was significantly decreased in differentiated NTera2 / CloneD1 cells. Conclusion: The model of NTera2 / CloneD1 cells differentiating into neurons was established. The expression of NRSF was significantly down-regulated in NTera2 / CloneD1 cells induced by NTera2 / CloneD1, which indicated that NTera2 / CloneD1 cells may be negatively regulated by NRSF down- Of neuron-specific protein initiation and upregulation, thereby achieving the directional differentiation of NTera2 / CloneD1 cells into neurons.