神经元限制性沉默因子在NTera2/CloneD1细胞向神经元分化模型中表达的检测

摘要

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/CloneDl细胞及经全反武维甲酸(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 neuronal differentiation from NTera2/CloneDl human embryonal carcinoma cell line, and to analyze the expression of neuron-restrictive silencer factor (NRSF) before and after induction of neuronal differentiation. Methods: NTera2/CloneDl cells were cultured in normal or in induction medium for 28 days. The morphological changes of NTera2/CloneD 1 cells after induction were observed under the microscope. Immunofluorescence staining method was used to analyze the expression of multipotent genes Nestin, Sox2 and typical neuronal markers NF-200, p-tubulin IE. RT-PCR and immunofluorescence staining were used to detect the expression changes of NRSF during neuronal induction. Results: Compared with clone-like growth of NTera2/ CloneDl cells under normal culture conditions, morphology of the NTera2/CloneDl-derived neurons changed, and presented typical neuronal morphology after the induction of retinoic acid(RA), arabinosylcytosine(AraC) and uri-dine. The immunofluorescence results showed that NTera2/CloneDl cells expressed nestin and Sox2, which were characteristic of the immature state of neuronal precursors. NTera2/CloneDl-derived neurons lose immunoreactivity for nestin and Sox2, and displayed immunoreactivity for NF-200, (5-tubulin III. The mRNA and protein expression levels of NRSF were significantly reduced in NTera2/CloneDl cells induced by RA, AraC and undine. Conclu-sion: We have successfully established the model of neuronal differentiation from NTera2/CloneDl cells. The expression of NRSF was significantly decreased and even diappeared in NTera2/CloneDl-derived neurons. These results indicate that RA, AraC and uridine may decrease the expression of NRSF and then initiate the expression of NRSF target genes.