BHLH Transcription Factors Regulate Electric Fields-induced Neuronal Differentiation

Objection:To investigate the expression patterns of bHLH transcription factors Hes1,Ascl1 and Oligo2 during neuronal differentiation induced by physiological electric fields(EFs).Materials and Methods:The Neural stem cells(NSCs)derived from embryonic mice brain were studied and divided into three groups: NSCs(Neurospheres),EFs(NSCs with EF-stimulation)and no EFs(NSCs that no EF-stimulation).NSCs was stimulated by EFs of150 m V/mm.RT-PCR,Western-blot and immunofluorescence chemical staining were used to detect the expression patterns of Hes1,Ascl1 and Oligo2 at m RNA and protein levels over time in the process of neuronal differentiation with EF-stimulation.Results:1.In vitro cultured NSCs,under the action of mitogenic factors b FGF/EGF,divide and proliferate to form Neurospheres.In the proliferative state,NSCs highly expressed Hes1,while Ascl1 and Oligo2 were weakly expressed.2.When b FGF/EGF was removed,NSCs stopped proliferating and entered the differentiation process,differentiating into neurons,astrocytes and oligodendrocytes respectively.In the absence of physiological electric field stimulation,most of these differentiated cells differentiated into astrocytes(45.80% ± 2.88%)and less differentiated into neurons(11.75% ± 1.76%).3.Under 150 m V/mm electric field stimulation,the proportion of Tuj1-positive neurons could reach 79.0% ± 3.35%.At the same time,the expression intensity of Ascl1 was also significantly increased,and the m RNA and protein expression levels increased accumulated with the extension of electric field treatment time,and reached the peak at 24 h of physiological electric field treatment.4.The expressions of Hes1 and Oligo2 showed no cumulative increase under the action of physiological electric field.The expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level.Oligo2 showed irregular changes in low expression level.Conclusions:1.Electric fields induced neuronal differentiation of neural stem cells.2.Electrical stimulation promotes the expression and accumulation of Ascl1 in NSCs;3.The stimulation of EFs promotes the continuous accumulation of Ascl1 expression and inhibits the expression of Hes1,thereby determining the differentiation of cells into neurons.