Effects Of Folate On The MRNA And Protein Expression Of Notch And MAPK Signal Pathway Of Neural Stem Cells

Objective To study the proliferation and differentiation effects of folate on neural stem cells(NSCs)via Notch and MAPK signal pathway in fetal rats and newborn rats in vitro.Methods NSCs of fetal SD rats from E14d to E16d and 24h newborn rats were cultured.In the normal condition,NSCs were divided into four groups which were control group,folate-deficiency group supplemented by methotrexate for 0.4μg/ml in media,folate-low group supplemented by folate for 4μg/ml in media,folate-high group supplemented by folate for 40μg/ml in media.When cultured with the inhibitor U0126 of MAPK pathway,NSCs were also divided into four groups which were control group,U0126 group supplemented by U0126 for 10μmol/L in media, folate-low group supplemented by U0126 for 10μmol/L and folate for 4μg/ml in media,folate-high group supplemented by U0126 for 10μmol/L and folate for 40μg/ml in media.After proliferation of NSCs had lasted for 6d,half of the cells were collected and the left were cultured for another 6d by adding FBS in order to promote NSCs to differentiate.Identification of the production of RT-PCR was via clone, enzyme digestion,sequence.The levels of related mRNA expression in MAPK signal pathway(ERK1,ERK2)were detected by SYBR Green I-PCR method.The levels of some kinds of proteins expression in fetal and newborn rats were detected by western blot method.Results 1.The result of general RT-PCR showed the production of PCR via clone,enzyme digest,sequence were specific and corresponded with anticipation.2.The SYBR Green I-PCR showed as follows when NSCs were cultured with inhibitor U0126 of MAPK signal pathway:There was no significant difference among the four groups although some groups supplemented by folate in NSCs of fetal rats(P＞0.05);There was a significant difference among some groups in NSCs of newborn rats(P＜0.05),but the results could not show the effect of folate.3.The western blot method showed when NSCs were cultured in normal condition and in the condition with inhibitor U0126 of MAPK signal pathway:(1) Notch pathway Folate could enhance the levels of Notch1 and HES5 proteins in proliferation in NSCs(P＜0.05);Folate could decline the levels of Neurogeninl and enhance the levels of HES5 in late differentiation period in NSCs(P＜0.05).(2) MAPK pathway No matter in proliferation or in differentiation,there was no significant difference among the four groups of the effect of folate on NSCs in fetal or newborn rats.Conclusions 1.When cultured in normal condition,folate could enhance the levels of Notch1 and HES5 proteins of Notch pathway to promote proliferation of fetal and newborn NSCs in proliferation period;Folate could enhance the levels of HES5 protein and decline the levels of Neurogeninl protein to promote fetal and newborn NSCs to differentiate into astrocytes in late differentiation period.2.When cultured with the inhibitor U0126 of MAPK pathway,the effect of folate on ERK1/2 mRNA in proliferation of NSCs in fetal and newborn rats was not significant;the effect of folate on the levels of pERK1/2 was not significant either, but there was a trend that folate could promote the levels of pERK1/2.3.No matter in the normal cultured condition or the condition with inhibitor U0126,the effect of folate on the levels of ERK1/2 mRNA and some proteins in NSCs of fetal and newborn rats was not significant.Therefore,folate could enhance the levels of some mRNA and proteins of Notch and MAPK signal pathway to promote proliferation and differentiation of NSCs in fetal and newborn NSCs,and this research will explore a new way of treat on CNS injury and neural degenerative disease.