The Influence Of Phenobarbital Sodium In ACSF On Proliferation Of The Cultured NSCs

Objective To establish the methods of culturing neural stem cells(NSCs)from newborn rat brain,observe the effects of artificial cerebrospinal fluid(ACSF)and phenobarbital sodium of different concentrations on differentiation of neural stem cells from neonatal rats.Methods NSCs were isolated from cerebrum of SD neonatal rats and cultured on the serum-free medium.The property of the cells was identified with Nestin which is one special sign for NSCs by immunocytochemistry medthod.BrdU was used to show the proliferation of the cells.The effects of ACSF on the cell survival rate were estimated by MTT reduction assay. The effects of different concentrations of Phenobarbital Sodium on the differentiation of NSCs were detected by immunocytochemistry.Scanning electron microscope was used to observe the neurons from differentiation.Results(1)The cultured NSCs showed multipotential differentiation into neurons and glial cells and the ability of self-renewing.The neurosphere could express Nestin and Bromodeoxyuridine(BrdU).The property of NSCs was stable after subculture.(2)MTT showed that 40%and higher concentration ACSF could significantly decrease the survival rate of neural stem cells(P＜0.05).(3)The results of immunocytochemistry indicated that the increase in concentration of phenobarbital sodium could decrease the ratio of MAP-2-positive cells and increase the ratio of GFAP-positive cells of differentiated cells as compared with control group.When the concentration of Phenobarbital Sodium is 10μg/ml,the ratio of MAP 2-positive cells was significantly decreased(P<0.01);When the concentration of Phenobarbital Sodium is 25μg/ml,the ratio of GFAP-positive cells was significantly increased(P＜0.01).(4) The neuron from experimental group had not the essential structures of the neuron functions by Scanning electron microscope.Conclusion The cultured NSCs express Nestin and have the ability of self-renewing and multi-directional differentiation.Phenobarbital sodium in ACSF inhibits the ratio of neurons and promotes the ratio of astrocytes simultaneously,and could degrade the differentiation of NSCs into normal neurons.