OBJECTIVE: To investigate the role of N-[N-(3,5-Difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretases inhibitor, on the proliferation and differentiation of neural stem cells (NSCs). METHODS: NSCs were isolated from Sprague-Dawley rat brain, cultured, and treated with DAPT for 6 weeks. Cell counting was conducted every 24 h. CCK8 assay was used to draw the growth curve. Immunofluorescence staining was performed to observe the proportions of beta-tubulin III positive cells (neurons), glial fibrillary acidic protein (GFAP) positive cells (astrocytes), and 2',3'-cyclic nucleotide3' phosphohydrolase (CNPase) positive cells (oligodendrocytes). RT-PCR was employed to assay the mRNA expression of RBP-Jk and Hes1 genes, downstream genes of the Notch pathway. RESULTS: Cell counting and CCK-8 assay showed that DAPT reduced the rate of NSC proliferation. Addition of DAPT altered NSC differentiation in vitro, percentage of The proportions neurons of the DAPT group was (13.84 +/- 1.22)%, significantly higher than that of the control group [(3.7 +/- 1.04)% , P <0.01], the proportion of the oligodendrocytes of the DAPT group was (14.75 +/- 1.58)%, significantly higher than that pf the control group [(4.8 +/- 1.22)%, P < 0.01]. However, the proportion of astrocytes of the DAPT group was (63.41 +/- 1.20)%, significantly lower than that of the control group [ (82.84 +/- 3.68)%, P <0.01]. The expression levels of RBP-Jk and Hes1 mRNA (RBP-Jk/GAPDH and Hesl/GAPDH) in the NSC treated with DAPT were 0.52 +/- 0.13 and 0.66 +/- 0.18 respectively, both significantly lower than those of the control group (0.28 +/- 0.06 and 0.16 +/- 0.08 respectively, both P <0.05). CONCLUSION: DAPT inhibits the NSC proliferation and alters the NSC committed differentiation. These effects are mediated via Notch signaling down regulation as a result of the inhibition of gamma-secretase.
OBJECTIVE: To investigate the role of N-[N-(3,5-Difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretases inhibitor, on the proliferation and differentiation of neural stem cells (NSCs). METHODS: NSCs were isolated from Sprague-Dawley rat brain, cultured, and treated with DAPT for 6 weeks. Cell counting was conducted every 24 h. CCK8 assay was used to draw the growth curve. Immunofluorescence staining was performed to observe the proportions of beta-tubulin III positive cells (neurons), glial fibrillary acidic protein (GFAP) positive cells (astrocytes), and 2',3'-cyclic nucleotide3' phosphohydrolase (CNPase) positive cells (oligodendrocytes). RT-PCR was employed to assay the mRNA expression of RBP-Jk and Hes1 genes, downstream genes of the Notch pathway. RESULTS: Cell counting and CCK-8 assay showed that DAPT reduced the rate of NSC proliferation. Addition of DAPT altered NSC differentiation in vitro, percentage of The proportions neurons of the DAPT group was (13.84 +/- 1.22)%, significantly higher than that of the control group [(3.7 +/- 1.04)% , P <0.01], the proportion of the oligodendrocytes of the DAPT group was (14.75 +/- 1.58)%, significantly higher than that pf the control group [(4.8 +/- 1.22)%, P < 0.01]. However, the proportion of astrocytes of the DAPT group was (63.41 +/- 1.20)%, significantly lower than that of the control group [ (82.84 +/- 3.68)%, P <0.01]. The expression levels of RBP-Jk and Hes1 mRNA (RBP-Jk/GAPDH and Hesl/GAPDH) in the NSC treated with DAPT were 0.52 +/- 0.13 and 0.66 +/- 0.18 respectively, both significantly lower than those of the control group (0.28 +/- 0.06 and 0.16 +/- 0.08 respectively, both P <0.05). CONCLUSION:DAPT inhibits the NSC proliferation and alters the NSC committed differentiation. These effects are mediated via Notch signaling down regulation as a result of the inhibition of gamma-secretase.