Effect Of Folate On Proliferation And Differentiation Of Neural Stem Cells By MAPK Signal Pathway

ObjectiveTo study the proliferation,differentiation and apoptosis effects of folate on neural stem cells(NSCs)via MAPK signal pathway in fetal rats and newborn rats in vitro.1.To establish the model of fetal rats and newborn rats in vitro;2.To observe the effect of folate on the proliferation of NSCs by MTT assay.3.To observe the effect of folate on the differentiation of NSCs by double-labeling immunofluorescence.4.To observe the effect of folate on the apoptosis of NSCs by FCM.Methods1.The NSCs from fetal rats(E14-16)and newborn rats(born in 24h)cerebral cortex were isolated and cultured by microdissection,mechanical blowing and serum-free suspension culture.2.Groups:NSCs were divided into four groups which were Control group(control), folate-deficiency group(MTX),folate-low group(Folate-L),folate-high group (Folate-H).In the U0126 cultured condition,NSCs were divided into four groups which were Control group(control),U0126 group(U0126),folate-low U0126 group(U0+LFA),folate-high U0126 group(U0126+HFA).After proliferation of NSCs had last for 6d,some of NSCs were collected and some of NSCs continued to culture for 6d by adding FBS in order to NSCs last for 6d to differentiate.3.MTT method was used to detect the growth of NSCs and the effect of folate on the proliferation of NSCs.4.The effect of folate on the differentiation of NSCs detect by double-labeling immunofluorescence.The effect of folate on the apoptosis and procession of cell cycle of NSCs was detected by FCM.5.The effect of folate on apoptosis rate of NSCs was detected by FCM.Results1.The cells that isolated and cultured by microdissection,mechanical blowing and serum-free suspension culture could be found nestin-positive cells.They possessed the ability of proliferation self-reproduction and can differentiation into neuron and astroglial cells being identified as neural stem cells. 2.Concentrations of folate in the range of 4,40μg/ml can promote the proliferation of NSCs of fetal rats and newborn rats in vitro particularly 40μg/ml folate promotes proliferation significantly.MTX inhabits the proliferation of fetal rats and newborn rats in vitro.In U0126 cultured condition,the differences between Control group and U0126 group were significant(P＜0.05).There was no difference between U0126+LFA group,U0126+HFA group and U0126 group.3.The results in respect to effect of folate on NSCs differentiation of fetal rats and newborn rats in vitro from double-label immunofluorescence technique are as follows:①Effects of folate on differentiation from NSCs to astrocytes in fetal rats:The differences between Folate-H group and the other three groups were significant (P＜0.05).Effects of folate on differentiation from NSCs to astrocytes in newborn rats:The differences between Folate-H group and control group,Folate-H group and MTX group,Folate-L group and MTX group were significant(P＜0.05).②Effects of folate on differentiation from NSCs to astrocytes in fetal rats after adding U0126:The differences between U0126 group and Control group were significant(P＜0.05).No significant difference was found among U0126 group, U0126+LFA group and U0126+HFA group(P＞0.05).Effects of folate on differentiation from NSCs to astrocytes in newborn rats after adding U0126:The differences between U0126 group and Control group were significant(P＜0.05). No significant difference was found among U0126 group,U0126+LFA group and U0126+HFA group(P＞0.05).③Effects of folate on differentiation from NSCs to neurons in fetal rats:No significant difference was found among the four groups(P＞0.05).Effects of folate on differentiation from NSCs to neurons in newborn rats:No significant difference was found among the four groups(P＞0.05).4.The effect of folate on the apoptosis of NSCs detect by FCM:During proliferation period,The differences between Control group and Folate-L group,Folate-H group were significant(P＜0.05),The differences between MTX group and Folate-L group,Folate-H group were significant(P＜0.05).During differentiation period,The differences between Control group and the other three groups were significant(P＜0.05),The differences between MTX group and the other three groups were significant(P＜0.05).Conclusion:1.This method is able to isolate and culture NSCs from fetal rats and newborn rats, these cells posses the ability of proliferation,self-reproduction and can be differentiated to neural and astrcyte,being identified as NSCs.2.Folate could promote proliferation of NSCs from fetal rats and newborn rat.After adding U0126,folate could not promote proliferation of NSCs from fetal rats and newborn rats,which suggests folate maybe promote proliferation of NSCs by MAPK signal pathway.3.Folate could promote differentiation of NSCs to astrocytes from fetal rats and newborn rat.After adding U0126,folate could not promote differentiation of NSCs to astrocytes from fetal rats and newborn rats,which suggests folate maybe promote proliferation of NSCs to astrocytes by MAPK signal pathway.4.Folate could inhabit apoptosis rate of NSCs during proliferation and nerve cell during differentiation period from fetal rats.