Astragalus Role In The Process Of In Vitro Differentiation Of Neural Stem Cells Research

Objective:The aim of this study was to investigate the effects of the Radix Astragali on the differentiation of the neural stem cells(NSCs) through the neural stem cells isolation and culture, and to detect the dynamic expression change of the relational gene—nestin,neuroD,ngn2 and Mash-1 in the process of neuronal differentiation of the neural stem cell,and to explore some inner molecular mechanism of the differentiation.To provided the experimental bases for the induced differentiation of neurons in vitro.Methods:(1) Cells were isolated from the 15d embryonic rat brain were cultured in the serum-free culture fluid.It was manifested that there were the morphological changes in the cells grown in the culture medium continuously can been seen under the inverted microscopy every day. Through the cell growth curve mapping to observe and verify that NSCs have the capacity of self-renewal and proliferation.Nestin is the markers of neural stem cell which were identified by chemoimmunity staining.(2) As the two generation neural stem cells were gained,they were separated into two groups:Radix Astragali were added into the experimental group,2mg/ml,20mg/ml,100mg/ml and 200mg/ml compared to the control group.To observe the morphological changes different concentrations of Radix Astragali on the effect of neurospheres and choice the best concentration of Radix Astragali for the next step.The cells we gained after added Radix Astragali for immunocytochemical detection of neuron-specific enolase(neuron specific enolase,NSE),glial fibrillary acidic protein(glial fibrillary acidic protein,GFAP) expression.Directly count the percentage of the neurons under the microscope.(3) The expression of the gene- nestin,neuroD,ngn2 and Mash-1 at the 1,3 and 5 days following the differentiation were detectd by RT-PCR.Results:(1) We have gained the neurospheres from the 15d embryonic rat brain.NSCs have the ability of self-renew,and can be separated or purified in vitro under some proper conditions. The neural stem cell can express the Nestin marker protein.(2) Cell calculation showed that the amount of the neurons differentiated from the neural stem cells were more remarkable in the Radix Astragali group.(3) In the process of Neural stem cell differentiation,Nestin continuous expression in the experimental group after 5 days.The expression of the three bHLH gene- neuroD,ngn2 and Mash-1 in the experimental group following differentiation increased more obviously than in the control group compared.During the differentiation of the neural stem cells,the three bHLH genes were all up-regulated,especially in dyal.Later,gradually weakened.Our experiment shows that there does promoted the bHLH.