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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.
To establish immortalized neuron-like cells that could be exposed to long-term environmental factors, human umbilical cord-derived mesenchymal stem cells were induced to differentiate into neuron-like cells using b FGF as a pre-inducer, BHA, DMSO and β-mercaptoethanol as an inducer , Observe the morphological changes of the cells, and detect the expression of Nestin, NSE, GFAP and MAP2 by cell immunofluorescence and RTPCR to judge whether they have the neuron-like cell specificity.All the cells that induce the success are passaged continuously and the single cell clone is isolated by the dilution method. The cells that can be passaged for more than 50 times were screened out, and the difference of Nestin and MAP2 gene expression was detected by RT-PCR to determine whether the neuron-like characteristics of the obtained monoclonal cells could be maintained. The results showed that the cell somatic cell bulge, immunofluorescence The positive expression of Nestin, NSE and GFAP protein showed that MSCs were successfully induced into neuron-like cells. Nestin gene expression was 1.32 times higher than that of the 11th passage, 8.85 times lower than that of the 50th passage, while the expression of MAP2 was 11 times Increased by 2.12 times and increased by 7.29 times at 50 times, indicating that it had differentiated from neural stem cells into neuron-like cells.The neuron-like cells obtained in this study could Continuous passage, and maintain neuronal-like cell characteristics, can be used as a cellular model to study the role of environmental factors in the chronic injury of the nervous system.