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Folic acid through MAPK signal pathway on neural stem cell proliferation and differentiation effect of

Author: TianZhiHong
Tutor: HuangGuoWei
School: Tianjin Medical University
Course: Nutrition and Food Hygiene
Keywords: Folate Neural stem cells Proliferation Differentiation Apoptosis MAPK
CLC: R329
Type: Master's thesis
Year: 2008
Downloads: 224
Quote: 0
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Abstract


Objective To study the folic acid on neural stem cells (neural stem cells, NSCs) proliferation, differentiation and apoptosis of U0126 and the role of folic acid added NSCs proliferation and differentiation effects and its mechanism. (1) Establishment of neural stem cells in vitro culture model. 2 by microscopic preliminary observations, MTT assay of folic acid on NSCs proliferation. 3 by confocal laser scanning fluorescence double staining, counting double positive cells detected NSCs differentiation. 4 by flow cytometry apoptosis effect of folic acid on NSCs. Methods 1.NSCs primary culture establishment: Play obtained by mechanical blowing embryonic rat, neonatal rat cerebral cortex NSCs, utilization factor containing bFGF and EGF in serum-free cell culture medium to obtain NSCs. 2 Test groups: selection of pregnant rats, neonatal rat cerebral cortex NSCs. Divided into four groups: ① normal control group (Control group); ② low dose of folic acid (Folate-L group); ③ high dose of folic acid group (Folate-H group); ④ folate deficiency group (MTX group). U0126 role, grouped as follows: ① normal control group (Control group); ② U0126 group (U0126); ③ U0126 low-dose folic acid group (U0126 low folic acid, U0 LFA); ④ U0126 high dose folic acid group (U0126 high folic acid, U0 HFA ). First collected serum-free medium for the application of some of the cells proliferating 6 d, and then add the remaining cells in serum makes NSCs differentiation of cultured 6 d, collected differentiated neural cells. 3 of folic acid on the proliferation of NSCs: preliminary observation under an inverted microscope and folic acid on NSCs proliferation, MTT was used to observe the impact of folic acid on NSCs proliferation and growth curve. 4 of folic acid on the differentiation of NSCs Study: preliminary observation under an inverted microscope effect of folic acid on differentiation of NSCs, confocal laser scanning fluorescence double staining of folic acid on the differentiation of NSCs into neurons and astrocytes proportional effect. 5 of folic acid on apoptosis of embryonic mouse NSCs: apoptosis rate by flow cytometry. Results 1. Mechanical blowing through play, including bFGF, EGF serum-free culture medium of NSCs express Nestin antigen, BrdU labeled showed proliferative capacity and can differentiate into expressing β-tubulin-Ⅲ neurons and GFAP star glue stromal cells, prove that the cultured cells were NSCs. 2 folic acid on cultured neonatal rat and embryonic NSCs proliferation studies: MTT results showed: 4μg/ml, 40μg/ml concentration of folic acid are to promote NSCs proliferation, 40μg/ml obvious, MTX inhibited embryonic rat and newborn rats the proliferation of NSCs; U0126 role in the control group U0126 group difference was statistically significant (P <0.05), U0 LFA and U0 HFA U0126 compared with no significant difference (P> 0.05). 3 folic acid on cultured neonatal rat and embryonic differentiation of NSCs confocal laser scanning fluorescence double staining results: ① folic acid on embryonic mouse NSCs into astrocytes differentiation ratio: Folate-H group and Control group comparison, Folate-H group and Folate-L group, Folate-H group and the MTX group, the difference was statistically significant (P <0.05). Folic acid in newborn rats NSCs into astrocytes differentiation ratio: Folate-H group and Control group, MTX group and Folate-L group, MTX group and Folate-H group, the difference was statistically significant (P < 0.05). ② U0126 under the effect of folic acid on embryonic mouse NSCs into astrocytes differentiation ratio: Control group with U0126 group, the difference was statistically significant (P <0.05), the remaining difference between the groups was not statistically significant (P> 0.05). Folic acid in newborn rats NSCs into astrocytes differentiation ratio: Control group with U0126 group, the difference was statistically significant (P <0.05), the remaining difference between the groups was not statistically significant (P> 0.05). ③ folic acid on embryonic mouse NSCs into neuronal differentiation ratio: comparison between groups, the difference was not statistically significant (P> 0.05). Folic acid on neonatal rat neuronal differentiation of NSCs proportion: comparison between groups, the difference was not statistically significant (P> 0.05). 4 Flow cytometry showed that apoptosis rate of embryonic mouse NSCs: appreciation of, Folate-L and Folate-H compared with the Control group, two groups of apoptosis were lower than Control group, the difference was statistically significant (P <0.05 ), Folate-L and Folate-H compared with MTX group, two groups of apoptosis were lower than MTX group, the difference was statistically significant (P <0.05). Differentiation, Control group and Folate-L group, Folate-H group and the MTX group among the three groups, Control group apoptosis rate in Folate-L group, Folate-H group, apoptosis rate, but lower than the MTX group withered death rate, the difference was statistically significant (P <0.05), Folate-L and Folate-H compared with MTX group, two groups of apoptosis were lower than MTX group, the difference was statistically significant (P <0.05). Conclusion 1 The pilot project is successful separation of the embryonic and neonatal rat brain NSCs, embryonic rats and newborn rats were having a neural stem cell basic features: the expression of neural stem cell-specific marker protein Nestin; divisive proliferation; can differentiate into neurons and glial cells. (2) to promote folic acid pregnant 14-16 d in vitro embryonic rats and newborn rats within 24 h NSCs proliferation effect of adding U0126, U0126 inhibition of folic acid-induced proliferation of NSCs, suggesting that folic acid may help promote the MAPK pathway through the proliferation of NSCs effect. 3 to promote folic acid pregnant 14-16 d in vitro embryonic rats and newborn rats within 24 h of NSCs into astrocytes differentiation, and differentiation into neurons is not obvious. Folic acid may promote through the MAPK signaling pathway in vitro E14-16 d embryonic rats and newborn rats within 24 h of NSCs into astrocytes differentiation. 4 folic acid can inhibit embryonic mouse NSCs apoptosis rate of value-added and differentiation of neural cell apoptosis.

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