Establishment An In Vitro Model Of Neural Cells Directional Differentiatied From Mouse Embryonic Stem Cell For Studying The Effects Of BFGF On Neural Development

Objective：To establish the experimental model of directional differentiation of embryonicstem cell （ESC） into neural cells in vitro, and discuss the effects of differentconcentration of basic fibroblast growth factor（bFGF） on the developmental processof mouse nerve.Methods:（1）EBs derived from ESC were induced directionally by5×10^-7mol/L ratinoicacid for two days and then medium was refreshed with ITS medium, then thecharacteristics of ES-derived neural cells were identificatied by using methods ofmorphological observation, immunofluorescence, and quantitative polymerase chainreaction.（2） ESC-derived neural cells were exposed to different concentration ofbFGF and the positive control drug valproic acid （VPA） for15days, neurotoxicity ofbFGF and VPA were determined by CCK8method.（3） Exposing ES-derived neuralcells to different concentration of bFGF for15days, the neural-specific geneexpression of ES-derived cells were detected by Q-PCR analysis, and theneural-specific protein expression were detected by western blotting analysis.（4）ES-derived neural cells were exposed to different concentration of bFGF for15days, the effects of bFGF on apoptosis were examined by flow cytometry and Hoechststaining.Results:（1） The model of directional differentiation of ESC into neural cells was successfullyestablished. The identification results of differentiation model of ESC into neural cellsby methods such as morphological observation, immunofluorescence, and quantitativePCR, indicated that ES-derived cells had neural properties, which could be used tostudy for evaluation of neural activities and toxicities of bFGF.（2） The model of directional differentiation of ESC into neural cells for evaluating theeffectiveness of neurodevelopmental toxicity of determinand object was proved to bevalid and effective. The model of neural cells differentiation from ES cells was established to detect neurodevelopmental toxicities and the effects of neural-specificgene expression of VPA exposure on ES-derived neural cells.The results indicated thatthe IC₅₀ of VPA in EST model and expression tendency of VPA on neural-specificgene were similar to the current research results, which indicated that theneural-induced model could be used to evaluate developmental neurotoxicity ofbFGF.（3） Effects of bFGF on the developmental process of mouse nerve. Using theestablished model of directional differentiation of ESC into neural cells to detecteffects of bFGF on neural developmental process. The results indicated that: bFGFIC₅₀=9.6μg/mL,0.1μg/mL bFGF could induce neuron differentiation and maturation,1μg/mL bFGF mainly accelerated the neural precursor cells growth, and improved theproliferation ability of mES-NPC, even the bFGF in high concentration of10μg/mL,still showed accelerating neural cells growth. Little obvious damage of ES-derivedneural cells treated by bFGF was perceived in the range of0.01-10μg/mL.（4） The apoptosis and oxidative injury which were induced by bFGF had not beenfond.Conclusions:In this study, we established the experimental model of directional differentiationof mouse ESCs into neural cells, and invistagated the effects of differentconcentration of bFGF on the developmental process of mouse nerve. The experimentresults showed that, within0.1-10μg/mL, bFGF mainly induced neuron differentiationand maturation, improved the proliferation ability of mES-NPC, and acceleratedneural cells growth. Little obvious damages of ES-derived neural cells treated bybFGF in commonly used concentrations and relative large concentrations weredetected, therefore, bFGF was relatively safe.