| Objectives:The purposes of the present study are:①to study the influence of cytomegalovirus infection on differentiation of neural stem cells (NSCs) in vitro.②to explore the effect of cytomegalovirus infection on the transcription of differentiation related genes in Wnt signal passway of NSCs.③to investigate the influence of cytomegalovirus infection on cyclins expression and cell cycle progression of NSC.Methods:①NSCs were isolated, cultured and identified, and it's differentiation potency were observed. NSCs were isolated from fetal brain of BALB/c mouse on day 13.5d of gestation and cultured in DMEM/F12 medium including EGF, bFGF and B27. Subcultured NSCs were identified by detecting nestin. NSCs were induced to differentiate in DMEM/F12 medium including 2% fetal bovine serum. Nestin, GFAP and NSE of cells were detected by immunofluorescence.②A differentiation culture model of infected NSCs was established. The NSCs infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively, were cultured in differentiation medium.③Morphological changes of infected NSCs and infection process were observed. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence (MOI=1) at 3d, 6d, 9d, 12d, 15d, 18d and 21d. The EA expression of MCMV was detected to observe the infection process.④The differentiated cells rate of infected NSCs was studied. Flow cytometry was employed to measure the rate changes of NSCs and its differentiated cells at 3d, 6d and 9d. ⑤The influence of MCMV on transcription of differentiation related genes in Wnt signal passway of NSCs in vitro were investigated. Real-time RT-PCR method was employed to measure the expression levels of the upperstream gene Wnt-3,Wnt-7a and the downstream gene Ngn-2,c-myc and CyclinD1 in Wnt signal pathway of NSCs at 0.5d, 1d, 2d, 3d, 4d and 5d.⑥The influence of MCMV infection on cyclins expression and cell cycle progression of NSCs in vitro were investigated. The NSCs were infected by MCMV with multiplicity of infection (MOI) equaled to 5, 1 and 0.1 respectively and cultured in DMEM/F12 medium including EGF, bFGF and B27. The dynamic changes of cyclinA, cyclinB1, cyclinD1, cyclinE and cell cycle progression of infected NSCs were detected at 1d, 2d, 3d, 4d, 5d and 6d.Results①NSCs were successfully isolated and cultured in vitro. NSCs could proliferate to form neurosphere and strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NeuN positive neurons or GFAP positive astrocytes.②The infected NSCs couldn't adhere to the wall and appear differentiation growth, but showed swelling gradually and disintegrated after differentiation culture. The nestin expression of the infected NSCs downregulated slowly and was higher than the control group (P<0.05). The expression of GFAP and NSE were lower than the control (P<0.05). The early antigen (EA) of MCMV could be always detected in the differentiated cells.③The rate of nestin positive cells was higher than the control group and the rate of GFAP and NSE positive cells were lower than it from 3d to 9d during differentiation culture(P<0.05).The changes of the infected groups were obvious following the MOI increased④The levels of Wnt-3 mRNA of infected groups decreased from 1d to 2d, obviously lower than that of normal control group (P<0.05), then increased but still not higher than the control. The levels of Wnt-7a and Ngn-2 mRNA were markedly lower than the control from 0.5d to 2d and at 1d, respectively (P<0.05) and both increased at 3d, but the general peak value were significantly lower than the control. The levels of c-myc mRNA were lower than the control from 1d to 4d (P<0.05). The levels of CyclinD1 mRNA were obviously lower than the control from 0.5d to 1d (P<0.05), and higher than it at 2d (MOI=5) and at 3d (MOI=5, 1, 0.1) (P<0.05). The expression change range of these genes increased following the MOI increase of MCMV.⑤The expression of cyclinA, cyclinB1, cyclinD1 and cyclinE of infected NSCs upregulated. The expression of cyclins of the infected NSCs with MOI=0.1 gradually increased and reached to the peak at 6d. The peak expression of cyclins of the infected NSCs with MOI=1 and 5 appeared at 4d and 3d post infection. The cell rate of G0/G1 phase decreased and the rate of S phase and G2/M phase increased. The infected NSCs migrated to and blocked at S phase and G2/M phase. These changes were more obvious following the MOI increased.Conclusions①MCMV could inhibit significantly NSCs differentiate into neurons and astrocytes and lead to the decrease of differentiated cells. The neurons and astrocytes during differentiation development period were both susceptible to MCMV.②MCMV could downregulate or interfere the expression of differentiation related genes Wnt-3, Wnt-7a, Ngn-2, c-myc and CyclinD1 in Wnt signal pathway of NSCs, which may be involved in the inhibitory effect of MCMV on NSCs differentiation.③The inhibitory effect of MCMV on NSCs differentiation and the influence of MCMV on the transcription of differentiation related gene of NSCs showed dose-dependent with MOI. MCMV could inhibit differentiation of NSCs by inhibiting the transcription of differentiation related gene, which may be one of primary causes of brain development disorders caused by congenital CMV infection. ④MCMV could influence the cell cycle processes of NSCs by upregulating expression of cyclinA, cyclinB1, cyclinD1, cyclinE and inducing NSCs enter into S phase from G0/G1 phase and appear S phase arrest and G2/M phase arrest. The effect showed certain dose dependence relationship with MOI, which may be major causes of MCMV inhibit the proliferation of NSCs.⑤The inhibitory effect of MCMV infection on differentiation and cell cycle progression of nerve stem cells maybe the important mechanisms of congenital encephalodysplasia induced by CMV. |
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