Differentiation into dopaminergic neurons from neonatal rat neural stem cells induced by ascorbic acid*,*,☆

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Studies regarding cell transplantation for Parkinson's disease focuses on searching appropriate cell lines and induction of abundant functional dopaminergic (DA) neuron from cells with differentiation potential.


To investigate the effects of ascorbic acid (AA) on the differentiation of neural stem cells (NSCs) into DA neurons in vitro.


In vitro observation of cytology. The experiment was performed at the Laboratory Center of Guangxi Medical University from January to December 2007.


A total of 30 Sprague Dawley rats (within 24 hours after birth) were provided by the Experimental Animal Center of Guangxi Medical University. AA was purchased from Sigma, USA.


The primary NSCs were isolated from whole brain of rats, suspended and cultured in serum-free medium in vitro at a density of 5×108/L (the second passage). Control cells were not given AA but only cultured in DMEM/F12 containing 10% fetal bovine serum and 2% B27; other cells were additionally induced with 50, 100, 200 μ mol/L for 10 days.


Tyrosine hydroxylase (TH) mRNA was detected by RT-PCR; NSCs and differentiation from NSCs to DA neurons were detected by immunocytochemistry.


Three AA groups had 795 bp amplified fragment, i.e. expressing TH mRNA. The neurospheres could proliferate and passage in vitro. Immunocytochemistry study indicated that the neurospheres were nestin-positive, had the capacity of self-renewing and proliferation, and could differentiate into multi-directions, specific antigens of neurons, astrocytes, and oligodendrocytes were expressed also. Compared with blank control group, AA increased the proportion of TH-positive cells (P < 0.05), in particular 100 and 200 μ mol/L was significantly higher than 50 μ mol/L (P < 0.05), while no significant difference was found between 100 and 200 μ mol/L AA (P > 0.05).


The NSCs isolated from neonatal rats show the capacity of self-renewing and proliferation, multi-differentiation, and expressing nestin antigen in vitro. AA (50-200 μ mol/L) can promote NSCs differentiate into DA neurons, and TH positive rate minimally changes when AA increases from 100 to 200 μ mol/L.

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