Effects Of Folate On The Mrna And Protein Expression Of Notch And Mapk Signal Pathway In The Proliferation And Differentiation 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 cultured condition, NSCs were divided into four groups which were normal control group (Normal control), folate-deficiency group (Folate-D) supplemented by methotrexate for 0.4μg/ml in media, folate-low group (Folate-L) supplemented by folate for 4μg/ml in media, folate-high group (Folate-H) supplemented by folate for 40μg/ml in media. In the hypoxia cultured condition, NSCs were divided into five groups which were normal control group (Normal control); and the four hypoxia groups, namely Hypoxia group (Hypoxia), folate-deficiency group (Hypoxia+Folate-D) supplemented by methotrexate for 0.4μg/ml in media, folate-low group (Hypoxia+Folate-L) supplemented by folate for 4μg/ml in media, folate-high group (Hypoxia+Folate-H) supplemented by folate for 40μg/ml in media. After proliferation of NSCs had last for 6d, some of NSCs were collected and some of NSCs continued to culture for 6d the proliferation by adding FBS in order NSCs last for 6d to differentiate. In hypoxia cultured condition, after proliferation of NSCs last for 6d, the hypoxia model was established, the hypoxia groups were reoxygen for 24h, and the whole cells were collected.1. Identification of the production of RT-PCR was via clone, enzyme digestion, sequence.2. The levels of related mRNA expression in Notch and MAPK signal pathway (Hesl, Hes5, ERK1, ERK2) were detected by SYBR Green I-PCR method.3. The levels of ERK1 and pERK2 expression in fetal rats in the normal cultured condition. were detected by western blot method. Results1. The results of general RT-PCR showed the production of PCR via clone, enzyme digest, sequence were specific and corresponded with anticipation.2. The results of SYBR Green I-PCR showed in the normal cultured condition as followed:①Folate could enhance the levels expression of Hesl and Hes5 mRNA in NSCs proliferation 6d in both fetal and newborn NSCs groups(P<0.05),but that in Folate-L was compared with that in Folate-L (P>0.05). Folate could decline the levels expression of Hesl and Hes5 mRNA in NSCs differentiation 6d in both fetal and newborn NSCs groups (P<0.05).②Folate couldn't regulate the levels of expression of ERK1 and ERK2 mRNA on proliferation and differentiation 6d in both fetal and newborn NSCs groups (P>0.05)3. The results of SYBR Green I-PCR showed in the hypoxia condition as followed:①Folate could enhance the levels of expression of Hesl and Hes5 mRNA in proliferation in NSCs of fetal rats (P<0.05). Folate couldn't regulate the levels of expression of Hesl mRNA on proliferation in newborn NSCs groups (P>0.05). Folate could enhance the levels of expression of Hes5 mRNA on proliferation in newborn NSCs groups (P<0.05).②Folate couldn't regulate the levels of expression of ERK1 and ERK2 mRNA on proliferation NSCs of fetal and newborn rats (P>0.05).4. Folate could enhance the levels of expression of pERKl and pERK2 on NSCs proliferation 6d (P<0.05), Folate couldn't regulate the levels of expression of pERKl and pERK2 on NSCs 6d differentiation (P>0.05).Conclusions1. In the normal cultured condition, folate could enhance the levels of expression of Hesl and Hes5 mRNA to promote proliferation of fetal and newborn NSCs on proliferation period; Folate could decline the levels of expression of Hesl and Hes5 mRNA to promote differentiation of fetal and newborn NSCs on differentiation period.2.In the normal cultured condition, no matter on proliferation or on differentiation, the effect of folate on the levels of expression of Hesl mRNA and Hes5 mRNA in fetal NSCs were as the same trend as that in newborn rats NSCs, but there was significance.3. In the hypoxia condition, the effect of folate on the levels of expression of Hesl and Hes5 mRNA in fetal NSCs were as the same trend as that in normal cultured condition. Folate could protect NSCs from hypoxia damage.4. No matter on the normal cultured condition or hypoxia condition, the effect of folate on the levels of expression of ERKl mRNA and ERK2 mRNA in NSCs of fetal and newborn rats were no significance.5. Folate could enhance the levels of expression of pERK1 and pERK2 to promote proliferation of fetal NSCs on proliferation period. Folate couldn't regulate the levels of expression of pERK1 and pERK2 on differentiation period.6. The dose of 40μg/ml was as same as 4μg/ml in the levels of expression of mRNA and protein.Therefore, folate had protective effects on CNS injury and neural degenerative disease by regulating related mRNA expression in Notch and MAPK signal pathway, and by regulating translation to promote NSCs proliferation and differentiation.