ABSTRACT
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.
Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Dipeptides/pharmacology , Neurons/drug effects , Stem Cells/drug effects , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Brain/cytology , Cells, Cultured , Fluorescent Antibody Technique , Gene Expression/drug effects , Glial Fibrillary Acidic Protein/metabolism , Homeodomain Proteins/genetics , Neurons/cytology , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Stem Cells/metabolism , Time Factors , Transcription Factor HES-1 , Tubulin/metabolismABSTRACT
A neural stem cell line (RNSC-FMU 1) from rat brain have been established successfully by isolating and culturing neural stem cells from newborn SD rat brain in vitro with free-serum medium and passaging with mechanical division. The cell line cultured can continuously generate in vitro for long-term and it is 21 months (>100 passages) so far. These cells keep the feature of neural stem cell and normal karyotype. These neural stem cells can be induced to differentiate into neurons, astrocytes and oligodendrocytes. The cells have an extensive self-renewal capacity; its doubling time of proliferation is about 20 h. The cells are also cryopreservable. Tumor formation is not observed in nude mice that explanted with the cells. This cell line is a good tool for research of neural stem cell.
