Effect Of EGF On BDNF-Induced Differentiation Of Adult Rat Hippocampus Neural Stem Cells Into Neurons

ObjectiveTo study the effect of epidermal growth factor (EGF) on brain-derived neurotrophic factor (BDNF) induced differentiation of adult rat hippocampus neural stem cells(NSCs) into neurons. To research the influence of Brain-derived neurotrophic factor on the proliferation and differentiation of neural stem cells(NSCs) from hippocampus of adult rats when the concentration of epidermal growth factor is 20ng/mL.MethodsThe DMEM/F12 media containing bFGF, EGF and B27 were used to cultivate and passage NSCs isolated from hippocampus of adult rats. Cell suspension was prepared and diluted when diameters of the fourth passaged cell spheres were 2μm. Monoclone was done by utilizing limiting dilution assay. Monoclonal cells were passaged and divided into two parts and done experiments respectively as follows:1. Identification of neural stem cellsImmunocytochemistry for Nestin was used to identify monoclonal NSCs. Immunocytochemistry for glial fibrillary acidic protein (GFAP) and neurone specific enolase (NSE) were done to identify astrocytes and neurons respectively a week after NSCs were differentiated.2. Differentiation of neural stem cells(1) Brain-derived neurotrophic factor (BDNF), epidermal growth factor(EGF) and fetal bovine serum were added in the medium. The concentration of EGF(20ng/mL) and fetal bovine serum (10%)was invariable. NSCs were divided for several groups that included 0ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, and 200ng/mL groups according to the doses of BDNF.(2) Growth factors and fetal bovine serum were added in the media. NSCs were divided into four groups: control, EGF, BDNF and EGF+BDNF groups by different growth factors added into the media. The media in control group were DMEM/F12 containing fetal bovine serum of 0.1 volume fraction. The media in the other three groups were added EGF, BDNF and EGF+BDNF respectively. The concentration of EGF was 20ng/mL and the concentration of BDNF was 50ng/mL.Immunocytochemistry for NSE was done a week later to count the positive cells.Results1. Identification of neural stem cellsThe cultured monoclonal spheres expressed Nestin, and expressed NSE and GFAP one week after induced differentiation.2. Influences of factors(1) The percentage of NSE positive cells differentiated from NSCs in 0ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL groups were 13.33±3.44%, 23.17±2.93%, 20.67±2.80%, 16.67±2.73%, 14.17±3.43% respectively. There was significant difference between groups that the concentration of BDNF was 50ng/mL, 100ng/mL and 0ng/mL group (P<0.05) in indication that NSCs differentiated to neurons.(2) The percentage of NSE positive cells differentiated from NSCs in control, EGF, BDNF and EGF+BDNF groups were 17.67+3.27%, 13.33±3.44%, 19.67±2.07%, 23.17±2.93% respectively. The proportion in the BDNF and EGF+BDNF groups were much higher than that in normal control and EGF groups with the highest in EGF+BDNF group.Conclusion1. EGF can improve the ability of BDNF in promoting the differentiation of adult rat NSCs into neurons.2. The proper concentration of BDNF that NSCs differentiated to neurons is 50ng/mL while the concentration of EGF was 20ng/mL.