一株用于研究环境因素暴露效应的神经样细胞

发布时间：2019-02-15 23:04

【摘要】：为建立一株可用于研究长期环境因素暴露的永生化神经样细胞,使用b FGF作为预诱导剂,BHA、DMSO和β-巯基乙醇作为诱导剂,诱导人脐带间充质干细胞向神经样细胞分化,观察细胞形态变化,并通过细胞免疫荧光和RTPCR检测Nestin、NSE、GFAP和MAP2的表达,判断是否具有神经样细胞特异性.将诱导成功的细胞连续传代,利用稀释法分离出单细胞克隆.筛选出可以连续传代50次以上的细胞,RT-PCR检测Nestin和MAP2基因表达差异,判断所获得的单克隆细胞的神经样特性是否能够维持.结果显示,诱导后的细胞胞体凸起,免疫荧光检测Nestin、NSE、GFAP蛋白表达均阳性,说明成功将MSCs诱导为神经样细胞,Nestin基因表达在传代11次时升高1.32倍,在50次时降低8.85倍,而MAP2基因表达在传代11次时升高2.12倍,在50次时升高7.29倍,说明其已经由神经干细胞分化为神经样细胞.本研究得到的神经样细胞单克隆可以连续传代,并且保持神经样细胞特性,可以作为检测环境因素对神经系统慢性损伤作用研究的细胞模型.
[Abstract]:In order to establish an immortalized neuron-like cell strain which can be used to study long-term environmental exposure, b FGF was used as preinducer and BHA,DMSO and 尾 -mercaptoethanol were used as inducers to induce human umbilical cord mesenchymal stem cells to differentiate into neural like cells. The changes of cell morphology were observed and the expression of Nestin,NSE,GFAP and MAP2 were detected by immunofluorescence and RTPCR. Single cell clones were isolated by dilution method. RT-PCR was used to detect the difference between Nestin and MAP2 gene expression and to determine whether the neuron-like characteristics of the obtained monoclonal cells could be maintained. The results showed that the cells were protruded, and the expression of Nestin,NSE,GFAP protein was positive by immunofluorescence, which indicated that MSCs was successfully induced into neuronal like cells, and the expression of Nestin gene increased 1.32 times after 11 passages. At 50 times, the expression of MAP2 gene decreased by 8.85 fold, while the expression of MAP2 gene increased 2.12 times at 11 times and 7.29 times at 50 times, which indicated that the expression of MAP2 gene had been differentiated from neural stem cells to neural like cells. The neuron-like cells obtained in this study can be subcultured continuously and maintain the characteristics of neuron-like cells. It can be used as a cell model for the study of the effects of environmental factors on the chronic injury of the nervous system.
【作者单位】：北京工业大学生命科学与生物工程学院;北京工业大学材料科学与工程学院;中国疾病预防控制中心病毒病预防控制所;
【基金】：北京市教委科技计划课题(KM201310005029) 973课题(2011-CB503705)资助~~
【分类号】：R114

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