Expression Of Notch Signal Molecules During Proliferation And Differentiation Of Neural Stem Cells

Objective: To isolate neural stem cells (NSCs) from neonatal rat cerebral cortex; to explore an improved method for dissociating neurospheres.To study the role of Notch signal pathway in the proliferation and differentiation of NSCs, through examining the expression of Notch1, PS1, RBP-Jk and HES1.With the aid of BMP-2 and EGF, to induce neuron differentiation and then to observe the expression of Notch pathway molecules.Methods:NSCs were cultured in serum-free media and were characterized with immunocytochemistry. Neurospheres were dissociated with plastic mesh pipette.During the proliferation and differentiation of NSCs, Notch pathway molecules were examined through RT-PCR and Western-blot.RT-PCR, Western-blot, immunocytochemistry and cell counting were employed to observe the neuron differentiation and Notch pathway molecules.Results:NSCs with the ability to proliferate and differentiate were isolated and cultured from neonatal rat cerebral cortex; Combined with digestion of trypsin, plastic mesh pipette could dissociate neurospheres with a high cell survival rate (92.1%).Data obtained from RT-PCR showed that, the expression of PS1 and RBP-Jk declined significantly on Day2; Notch1 and HES1 did not show any obvious changes during all the periods. Western-blot showed that the expression of PS1 declined on Day2, but not in the other periods. Compared with group EGF and Ctrl, the percentage of neurons in group BMP-2 was much higher; the percentage of neurons showed no difference between group EGF and Ctrl. Neither the mRNA level of Notch pathway molecules, nor the protein level of PS1 showed any difference among these 3 groups. ConclusionsThe cells isolated from neonatal rat cerebral cortex were NSCs which were able to proliferate and differentiate; with high survival rate, a plastic mesh pipette could dissociate neurospheres effectually.Notch signal pathway is essential for the NSCs in vitro to maintain proliferation. BMP-2 shows more influence on neuron differentiation than EGF and Ctrl; there is no relationship between Notch signaling and percentage of neurons.