Research Of Total Saponins Of Astragalus Promoting Exogenous Neural Stem Cells Differentiated Into Neurons And Its Mechanism

Objective1.Compared the effects of two culture-medium on the proliferation of neural stem cells originated from the cerebral cortex of neonatal SD rats,which could improve the culture method of neural stem cells.2.To observe the effects of total saponins of Astragalus（TSA）on neural stem cells（NSCs）differentiating to neuron in vitro and the best concentration,as well as the role of Wnt/β-catenin signaling pathway in it.Methods1.After separation cells of cerebral cortex,established control group using medium of Neural basal+B-27 and experimental group using medium of DMEM/F12+B-27+EGF+bFGF.The morphology of primary cells was observed by inverted microscope.Selected the third generation cells in each group,CCK-8 was used to count the cell early proliferation of and immunofluorescence was used to count the number of neurospheres during passage.Cell differentiation was induced in experimental group and identified the morphology of differentiated cells by Immunofluorescence.2.NSCs from the cerebral cortex of a neonatal rat were cultured and identified.The possible effective concentration of TSA was screened with Cell Counting Kit-8（CCK-8）.The third generation of NSCs was induced as normal group（without TSA）and various concentration of TSA groups for seven days.The expression of neuron specific protein（MAP-2）and astrocyte specific protein（GFAP）was detected with indirect immunofluorescence and Western blotting to detect the ratio of neuron and astrocyte.Then,the third generation of NSCs was induced as normal group（without TSA）,the best concentration of TSA group,TSA group and inhibitor ICG-001 group,and ICG-001group,for seven days,and the expression of Wnt3/3a,β-catenin and neurogenin 1（Ngn1）was detected with Western blottingResults1.The cellular form in experimental group was closer to the typical neurosphere than in the control group.The early stage of cell proliferation,the number of cells in the experimental group was significantly higher than that in the control group（P<0.05）.The passage stage,the number of neurospheres in the experimental group was significantly higher than that in the control group（P<0.05）.The cells in the experimental group had the ability to differentiate into neurons and glial cells which could be clearly labeled.2.TSA had a positive effect on the number of NSCs in the concentration of 1×10^-44 mmol/mL,1×10^-55 mmol/mL and 1×10^-66 mmol/mL（P<0.05）which could be used as a concentration for subsequent differentiation experiment group,and increased the proportion of neurons differentiated from NSCs,especially in the concentration of 1×10^-5mmol/mL（P<0.05）.TSA increased the protein expression of Wnt3/3a,β-catenin and Ngn1.Conclusion1.DMEM/F12+B-27+EGF+bFGF medium could better promote the proliferation of neural stem cells in neonatal SD rats than Neural basal+B-27 medium.NSCs cultured in this way have the good ability to differentiate.2.TSA could promote NSCs differentiating into neurons in vitro,which may associate with the activation of the Wnt/β-catenin signaling pathway and the expression of differentiation-promoting target protein Ngn1.TSA can do it best in the concentration about 1×10^-5mmol/mL...