| Objectives To investigate the influence of MCMV (murine cytomegalovirus) infection on the protein expression of upstream-differentiation-related target genes (Wnt-3, Wnt-7a) of Wnt signaling pathway in neural stem cells (NSCs) and explore the molecular mechanisms of fetal encephalodysplasia caused by congenital CMV infection.Methods⑴The isolation and culture of murine NSCs: NSCs were isolated from fetal brain of BALB/c mouse on day of 13.5 of gestation, washed with PBS, and filtered to make for single cell suspension, then cultured in DMEM/F12 medium supplemented with EGF (20ng/mL), bFGF (20ng/mL) and B27 (2%). NSCs of the third generation were used in the following experiments.⑵The establishment of the differentiation and culture model of NSCs infected by MCMV: The NSCs were infected by MCMV Smith strain with high, median and low multiplicity of infection(MOI) of 5, 1, and 0.1 respectively, then cultured in DMEM/F12 medium including two percent of fetal bovine serum (without EGF, bFGF and B27) to induce the differentiation. Normal NSCs of homochronous culture and differentiation without infection was as the negative control.⑶The influence of MCMV infection on the protein expression of the upstream-differentiation-related target genes(Wnt-3, Wnt-7a) of Wnt signaling pathway in NSCs : The dynamic protein expression of Wnt-3 and Wnt-7a in NSCs were measured by Western Blot assay at 0.5d, 1d, 2d, 3d, 4d and 5d after differentiation culture. All of the experiments were repeated thrice.Results⑴Murine NSCs were isolated and cultured in vitro successfully, and they could proliferate and duplicate with the appearance of neurosphere, and they also could be induced to differentiate when cultured in DMEM/F12 medium including two percent of fetal bovine serum (without bFGF, EGF and B27).⑵After MCMV infection, the adherence of NSCs were affected, the adherented nerosphere swelled and floated, and the marginal cells of the neurosphere dropped off, partial cells disintegrated. Such morphological change was more obvious with the MOI of virus increased.⑶The results of the Western blot assay: a. The expression of Wnt-3 protein of normal control showed the feature of two crest on day 2 and day 5 after differentiation, and the second crest was higher than the first. Although that of infection group also had a crest on 2d, only the subgroup of MOI=0.1 showed the second crest, which was obviously lower than the first. In MOI=1 and MOI=5 subgroup, the expression of Wnt-3 reduced gradually after 2d with absence of the second crest. The levels of Wnt-3 protein of the infection groups were obviously lower than those of normal control at all time points except 2d of the MOI=0.1 group (P<0.05); the levels of Wnt-3 of MOI=1 subgroup and MOI=5 subgroup were significantly lower than those of MOI=0.1 on 0.5d, 2d and 1d, 2d, 5d respectively(P<0.05); b. In normal control, the protein expression of Wnt-7a increased first, then decreased, and reached the peak on day 1 after differentiation. And that of the MOI=0.1 subgroup were similar. However, in MOI=1 and 5 subgroup, the level of Wnt-7a was obviously lower than that of normal control and MOI=0.1 subgroup on 0.5d and 1d (P<0.05), the peak of Wnt-7a expression delayed to day 3 after differentiation, and the peak of MOI=1 subgroup was significantly lower than that of normal control(P<0.05).Conclusions1) MCMV inhibited the protein expression of Wnt-3 in differentiated NSCs, which was more apparent with MOI increasing and differentiation proceeding. MCMV inhibited the protein expression of Wnt-7a in earlier period of differentiation (0.5d～1d), and delayed their peak, which also showed dose-dependence with MOI.2) MCMV could inhibit the protein expression of upstream-differentiation-related target genes (Wnt-3, Wnt-7a) of Wnt signaling pathway in NSCs, which may be one of the important mechanisms causing the impairment of fetal brain through interfering the proliferation and differentiation of NSCs.
|
PDF Full Text Request