ABSTRACT
Objective To study any effect of repetitive magnetic stimulation (rMS) on the differentiation and apoptosis of rat neural stem cells in vitro.Methods The bilateral hippocampus of a 3-day old Sprague-Dawley rat was used to culture neural stem cells (NSCs) in vitro.P2 NSCs were differentiated to neurons or astrocytes in differentiation medium and then divided into a control group in which the NSCs differentiated naturally,and an rMS group in which 1000 impulses/day of rMS were applied at 10 Hz once a day for 7 days at 50％ of maximum output.One hour after the last stimulation,immunofluorescence was used to analyze the ratio of neurons and astrocytes,and Western blotting was employed to evaluate the expression of glial fibrillary acidic protein (GFAP),β-Ⅲ tubulin and brain-derived neurotrophic factor (BDNF).NSCs which had differentiated for 7 days without stimulation were then selected and divided into an apoptosis group and an apoptosis+rMS group.The same rMS protocol was applied to the latter group 1h after the apoptosis,and 4h later flow cytometry (anexin V-FITC) was employed to evaluate the apoptosis ratio.Bcl-2,Bax and caspase-3 protein expression were analyzed using Western blotting.Results There were no significant differences between the control and rMS groups in the proportion of NSCs differentiating to neurons or in β-Ⅲ tubulin,GFAP or BDNF protein expression.The cell apoptosis rate of the apoptosis+rMS group was significant lower than in the apoptosis group.Caspase-3,Bcl-2 and Bax protein expression were also significantly different between the two groups.Conclusion rMS at 10Hz for 7 days has no effect on the differentiation of NSCs,but it has a protective effect on neural cells and decreases the apoptosis rate.
ABSTRACT
Objective To study the mechanism of rats' neural stem cells (NSCs) proliferation in vitro after repetitive magnetic stimulation (rMS).Metbods The bilateral hippocampus of neonatal Sprague-Dawley rats (＜3 d) was taken out to culture NSCs in vitro.The OD value was evaluated with Cell Counting Kit-8 (cck-8) and cell growth curve was generated.The NSCs cultured were divided into a control group and an rMS group.rMS (3 days,once per day) was applied on p2 NSCs at 10 Hz,50％ machine output and 200 pulses per day.One hour after the last rMS,the cck-8 was used to test the cell proliferation,and the western blotting was applied to detect the protein expression of c-fos and p-CREB.Results The nestin fluorescent staining of p2 neurospheres was proved to be neural stem cells.The growth curve indicated that their viability reached the peak on the third day.The OD value in the rMS group (0.309 ± 0.043) showed a significant difference (P ＜ 0.05) after rMS compared with the control group (0.256± 0.043).So did the c-fos and p-CREB protein expression between the two groups (P ＜ 0.01).Conclusion The rMS at 10 Hz can promote rats' NSCs proliferation in vitro,which may be related to the increased expression of p-CREB and c-fos after rMS.
ABSTRACT
Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (P<0.05 or 0.01 for all). The expression levels of p21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (P<0.05 or 0.01 for all). In conclusion, our findings suggested that rMS enhances the NSCs proliferation in vitro in a dose-dependent manner and miR-106b/p21/cdks/cyclins pathway was involved in the process.