Proliferation And Differentiation Of Neural Stem Cells Are Selectively Regulated By Knockout Of CyclinD1

Objective To isolate and identify neural stem cells (NSCs) from subventricular zone(SVZ) and hippocampus of neonatal mouse, and to observe the characteristics of proliferation and differentiation of NSCs.Methods Neurobasal medium+B27 a basic medium was utilized to isolate and incubate NSCs from SVZ and hippocampus of neonatal mouse. Fluorescence immunocytochemistry was applied to detect the antigen of nestin, TUJI and GFAP.Results The cells isolated from SVZ and hippocampus of neonatal mouse possessed the ability of proliferation and self-renew. Neurobasal medium+B27 as a basic medium could culcured the cell clone sphere. Nestin positive cells could be found in the cell clone sphere. Cells induced to differentiate could express antigen of TUJI and GFAP. These differentiated cells also appeared cellular specific morphologies. Interestingly, cells double labeled for (3-tubulin and GFAP were observed in neurosphere medium supplemented with 1% FBS (Gibco) in the absence of mitogens. The percentages of the cells positive for both P-tubulin and GFAP show temporal decrease along with the differentiation time(77.33±3.79% at day 7,23.67±4.16%, at day 14 and 14.67±3.06% at day 20).Conclusion The NSCs were successfully isolate from SVZ and hippocampus of neonatal mouse. The cells possess the ability of self-renew, proliferation, and differentiation. AIM:To explore the effects of Knockout of cyclinDl on the cell cycle and apoptosis of neural stem cells (NSC) in mice.METHODS:Use cyclin D1 gene knock-out mouse, observe the effect of cyclin D1 gene knock-out on on the cell cycle and apoptosis of neural stem cells (NSC) in mice by TUNEL and flow cytometry.RESULTS:Knockout of cyclinD1 induced the cultured neural stem cells arrested at G0/G1-phase as detected by flow cytometry. CyclinD1-/- mice, strong TUNEL staining was detected in most cells and the percentages of apoptotic cells was much higher than that of CyclinDl+/+mice (55.00±6.24 versus 18.00±3.61).CONCLUSION:Knockout of cyclinDl induced the cultured neural stem cells arrested at G0/G1-phase, at the same time, Cyclin D1 knockout led to the apoptosis of NSCs in vitro. Aim We further determined whether inhibition of the cell cycle by cyclinDl gene knockout could affect the differentiation of neural stem cells.Methods Specific mediums were used to induce NSCs differentiate into neurons or astrocytes selectively. To induce NSCs differentiate into neurons, NSCs were cultured in N2 medium (Invitrogen) containing RA and forskolin; In order to stimulate NSCs differentiate into astrocytes, cells were cultured with BMP-2, LIF and FBS for 4 days. Half of the medium was replaced after 48 h of incubation in the respective defined medium. Cells were then fixed and stained forβ-tubulin-Ⅲ, GFAP by immunocytochemistry.Results In the N2 medium containing BMP-2 and LIF for astrocyte differentiation, there was no obvious difference in the percentage of GFAP+ cells between the NSCs cultures from cyclinD1+/+ and CyclinDl+/- mice. However, a marked and significant reduction of the percentage of GFAP+ cells was found in cyclinD1-/- mice(P< 0.01). Nevertheless, there was no significant difference among the percentages of P-tubulin-Ⅲ(+) cells in the NSCs cultures from cyclinD1+/+, CyclinD1+/-, cyclinD1-/ mice.Conclusion Knockout of cyclinDl inhibited neural stem cell differentiate into astrocyte but without affecting the differentiation into neurons AIM:To assess the effect of cyclinDl on proliferation of neural stem cell proliferation in DG and SVZ zone in vivo.Methods:The 4-to 5-week-old mice were given an intraperitoneal (i.p.) injection of BrdU dissolved in 0.9% NaCl (50 mg/Kg body weight). Cyclin D1+/+, Cyclin D1+/-and Cyclin D1-/- mice were subdivided into three groups that were separately sacrificed on days 7 after BrdU injection. we examined the rate of 5-bromodeoxyuridine (BrdU,50mg/ml) positive cells in DG and SVZ cells by immunochemistry.Results:As compared with cyclinD1+/+ and CyclinD1+/- mice, cyclinD1-/- mice showed a significantly reduction of BrdU positive cells at both DG and SVZ zones (DG,16.67±1.76% in cyclinD1-/- mice versus 33.00±1.16% in wild type mice; P< 0.01). In SVZ zone, cyclinD1 knockout demonstrated much greater inhibition on cells positive for Brdu (31.67±2.03 (cyclinD1-/-) versus 92.00±4.36 (wild type); P< 0.01).Conclusion:Knockout of cyclinD1 inhibits neural stem cell proliferation in DG and SVZ zone.