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Objective To study the folic acid (folate) embryos, newborn Sprague-Dawley (SD) rats the proliferation and differentiation of neural stem cells (neural stem cells, NSCs), and to explore its mechanism. Methods pregnant 14d ~ 16d SD embryonic mice, and littermates within 24h newborn mouse whole brain, the NSCs original culture. Divided into four groups: under normal culture conditions, (3) low-dose folic acid group (Folate ① normal control group (Normal control); ② lack of folic acid group (Folate-D) culture medium given methotrexate dose 0.4μg/ml; -L) cultured the solution given folic acid dose 4μg/ml; ④ high doses of folic acid group (Folate-H), folic acid 40μg/ml. Under hypoxic culture conditions were divided into five groups: ① normal control group (Normal control); ② hypoxia model group (Hypoxia); ③ hypoxia folic acid deficient group (Hypoxia Folate-D) give methotrexate 0.4 μg / ml; ④ the anoxia low dose folic acid group (Hypoxia Folate-L) given folic acid dose 4μg/ml; ⑤ hypoxia high doses of folic acid group (Hypoxia Folate-H) given folic acid dose of 40μg/ml. The collected first application of serum-free medium for some of the cells in the proliferation 6d, and then the the remaining NSCs cells and serum to make differentiation, continue to cultivate 6d, collected differentiated neural cells. In addition, hypoxia conditions, NSCs proliferation 3d, and then create the hypoxia model reoxygenation 24h, and finally collected all the proliferative phase NSCs. A method of RT-PCR products were identified by cloning, restriction enzyme digestion and sequencing. Using real-time quantitative SYBR Green I-PCR method determination of NSCs Notch signaling pathway genes (Hes1, Hes5) and MAPK signaling pathway genes (ERK1, ERK2) mRNA expression. Using the Western blot method, the detection of normal cultured embryonic mouse under conditions proliferation / differentiation stage of NSCs pERK1 pERK2 protein content. 1. Ordinary RT-PCR amplification products were cloned, restriction enzyme digestion, sequencing, specificity amplified fragment with the expected results. 2. SYBR Green I-PCR results showed that, under normal culture conditions: ① proliferation 6d, folic acid increased embryonic mouse the newborn mouse NSCs Hes1, Hes5 mRNA expression (P lt; 0.05), but no difference between folic acid and low dose group statistically significant (P gt; 0.05). Differentiation 6d, folic acid reduce embryonic rat neonatal mouse NSCs Hes1, Hes5 mRNA expression (P lt; 0.05). ② proliferation 6d, folic acid supplementation on embryonic mice, newborn mouse NSCs of ERK1, ERK2 mRNA expression had no effect, but Folate-D group than in the other groups (P lt; 0.05). Differentiation 6d, Folate-H of embryonic rat ERK2 mRNA expression is higher than the other groups (P lt; 0.05), folic acid supplementation on embryonic mice, newborn mouse NSCs of ERK1, ERK2 mRNA expression had no effect. 3. SYBR Green I-PCR results show that folic acid on the proliferation of newborn rats NSCs Hes1 mRNA under hypoxia conditions: 1 Folic acid supplementation can increase the proliferation of embryonic mouse hypoxia groups Hes1 Hes5mRNA expression (P lt; 0.05); expression had no effect (P gt; 0.05), folic acid can increase the proliferative phase of newborn mouse NSCs Hes5 mRNA expression (P lt; 0.05). ② folic acid murine proliferative stage embryos, newborn mouse NSCs of ERK1 and ERK2 mRNA expression of affect (P gt; 0.05). 4. Western blot test results under normal culture conditions embryonic mouse NSCs proliferation / differentiation midterm pERK1 pERK2 protein expression. Proliferation 6d, folic acid can increase the embryonic mouse the NSCs pERK1, pERK2 protein expression. Differentiation 6d, folate embryonic mouse NSCs pERK1 pERK2 protein expression had no effect. Conclusion 1 under normal culture conditions, folic acid, possibly through embryonic mice raised the proliferative phase, the newborn mouse NSCs Notch signaling pathway Hes1, Hes5 mRNA expression promote NSCs proliferation; rat by down-regulating the differentiation stage embryos, newborn rats NSCs Hes, Hes5 gene mRNA expression and promote the differentiation of NSCs. Under normal culture conditions, in terms of proliferation or differentiation period of folic acid on embryonic rat neonatal mouse NSCs Hes1 Hes5 mRNA expression regulation trends are basically the same, but there are some differences. Hypoxic culture conditions, the trend of folic acid on the expression of related gene mRNA and protein expression in embryonic mice, newborn rats proliferation of Notch and MAPK signaling pathways trends with normal culture conditions. Folic acid may change by regulating gene mRNA, improve the damage caused by hypoxia on NSCs. 4 under hypoxia conditions under normal culture conditions, the folic acid on proliferation, differentiation stage of embryonic mouse / newborn rats NSCs MAPK signal pathway ERK1, ERK2 gene mRNA regulation is not obvious. 5. Folic acid can regulate the proliferation of embryonic mouse NSCs MAPK pathway pERK1 the pERK2 protein expression, promote NSCs proliferation; folic acid on the differentiation stage embryos mice of NSCs pERK1 pERK2 protein expression had no effect. Whether under normal culture conditions, or hypoxia conditions, the folic acid 40μg/ml dose groups with 4μg/ml of dose group of embryonic mice, newborn mouse NSCs proliferation, differentiation, Notch and MAPK pathways in the regulation of gene and protein The basic role. , Folic acid may affect the mRNA expression of Notch and MAPK signaling pathways, thereby affecting the translation of each gene, so as to promote the proliferation of NSCs differentiation play a certain role in the prevention and treatment of nervous system injuries and neurodegenerative diseases.
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