The Research On Neural Stem Cells Differentiate Into Dopaminergic Neurons In Vitro

Objective:To investigate the neural stem cells(NSCs)isolation,culture and expansion.To provide the experimental bases for the directional differentiation of dopaminergic(DA)neurons,we studied the effects of ascorbic acid(AA)and glial cell line-derived neurotrophic factor(GDNF)on the differentiation of NSCs into DA neurons in vitro.Methods:(1)Isolated the primary NSCs from whole brain of neonatal Sprague Dawley rats(within 24h after birth),the cells were suspended and cultured in serum-free medium in vitro.The cell morphology was observed with inverted phase-contrast microscope.Ploting the growth curve to observe the capacity of self-renewing and proliferation of NSCs.Immunocytochemistry was performed to detect the expression of neuroepithelial stem cell protein(Nestin), neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),and cyclic nucleotide phosphohydrolase(CNP).(2)The P 2(passage 2)NSCs were seeded in the differentiation medium supplemented with different concentration of AA or 10ng/ml GDNF or 100μmol/l AA+10ng/ml GDNF respectively.After these cells were induced for 10 days,the specific antigens of DA neurons including tyrosine hydroxylase(TH)and dopamine transporter(DAT)were detected by Immunocytochemistry.TH mRNA was detected by RT-PCR.Results:(1)The proliferating cells isolated from neonatal Sprague Dawley rats grew into floating neurospheres in the serum-free medium.The neurospheres had the capacities of self-renewing and expressing Nestin.The cells differentiated from these neurospheres could express the specific antigens of neurons,astrocytes,and oligodendrocytes.(2)Each group of different concentration of AA,10ng/ml GDNF and 100μmol/l AA+ 10ng/ml GDNF could increase the proportion of NSCs differentiated into TH-positive cells compared with the control group(p＜0.05).When treated with 100μmol/l AA +10ng/ml GDNF,the proportion of NSCs differentiated into TH-positive cells was significantly higher than 100μmol/l AA or 10ng/ml GDNF group alone(p＜0.05).Each group could detect the expression of TH mRNA.Conclusions:(1)The NSCs were isolated from neonatal rats successfully. In vitro,they showed the capacities of self-renewing and proliferation, pluripotentiality of differentiation,and expressed the Nestin antigen.(2)In our studies,each group of AA could promote the differentiation of NSCs into DA neurons.(3)GDNF could promote the differentiation of NSCs into DA neurons, when treated with GDNF + AA,the effect was stronger than AA or GDNF group alone.