| Objective To identify the upstream molecular mechanism of Brn-4in promoting neuronal differentiation of hippocampal neural stem cells (NSCs) through investigating the effect of IGF-1on the expression of Brn-4in hippocampal neural stem cells.Methods The in vitro cultured hippocampal neural stem cells were treated with insulin growth factor (IGF-1).1. MAP2immunofluorescence staining was used to determine the neurons after differentiation and Brn-4expressed;2. Brn-4immunostaining was used to determine its expression in hippocampal NSCs and Real-time PCR was adapted to detect the levels of Brn-4mRNA at different time points, from which we get the optimal treatment time of IGF-1;3. The hippocampal NSCs were pretreated with signal molecule inhibitors (PI3K:LY294002, MAPK:PD98059) before IGF-1treatment. And Western blot and Real-time PCR were used to detect the levels of Brn-4protein and mRNA.Results After induced to differentiation with IGF-1more of hippocampal NSCs differentiated into MAP2positive neurons, which were also immunoreactive to Brn-4, than the control group (p<0.05); Real-time PCR indicated that the expression of Brn-4in the hippocampal NSCs increased in a time manner and reached the peak at24hours after IGF-1treatment; Pretreatment with PI3K inhibitors LY294002markedly reduced the IGF-1-induced increase of Brn-4in hippocampal NSCs by using Western blot and Real-time PCR (p<0.05), which showed no significant difference with the control group (p>0.05); However, the cell group treated sequently with MAPK channel inhibitors PD98059and IGF-1did not show the obvious decrease of Brn4expression compared with IGF-1treatment group (p>0.05).Conclusions IGF-1can promote hippocampal NSCs to differentiate into neurons and at the same time increase the expression of Brn-4which has been proved to be involved in neuronal differentiation of hippocampal NSCs. PI3K pathway mediated IGF-1-induced Brn-4expression in the hippocampal neural stem cells. |
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