The Effect Of γ-secretase Inhibitor-DAPT On Differentiation And Proliferation Of Neural Stem Cells In Rats

Objective:To investigate the effect of DAPT (one kind ofγ-secretase inhibitors) on differentiation and proliferation of cultured neural stem cells from rat in vitro;To examine the expression of genes of Notch signal pathway and know the proteins that associated with Notch signal and other charaters of neural stem cells; to reveal the possible mechanism about how DAPT regulate the differentiation and proliferation of neural stem cells.Methods:1. NSCs were isolated from postnatal day 2~3 rat cerebral cortex using serum-free neurospheres method, and NSCs cultured for 4 weeks were identified by BrdU incorporation, with selective marker nestin, and the abilities of differentiation into neurons (β-tubulinⅢpositive), astrocyrtes (GFAP positive) and oligodendrocytes (CNPase positive).2. NSCs cultured for 4 weeks were induced to differentiate with DMEM/F12 with 2% B27 and 200nmol/L DAPT, the genes of Notch signal pathway, such as RBP-Jk and HES1, were examined by RT-PCR.3. DAPT was used to inhibit the proliferation of NSCs cultured for 4 weeks. Growth rate of NSCs was determined by cytometry and CCK-8.4. NSCs cultured in 4 weeks were induced to differentiate by DMEM/F12 with 1%FCS+2%B27 and 200nmol/L DAPT for 3 days, then immunofluoresence staining was applied to investigate the percentages of neurons, astrocyrtes and oligodendrocytes.5. The two-dimensional gel electrophoresis and liquid chromatograpy/mass selective detector electrospray ionization mass spectrometry(LC/MSD-ESI MS) were used...