ABSTRACT
AIM:To remark the effect of Qingguang'an Ⅱ on expression of PAX6, Ngn1, and Ngn2 mRNA of rats with chronic high intraocular pressure.METHODS:Totally 40 male SD rats were randomly divided into 6 groups, that was:A:blank group, B:model group, C:Qingguang'an Ⅱ low dose group, D:Qingguang'an Ⅱ moderate dose group, E:Qingguang'an Ⅱ high dose group, F:Yimaikang disket group.B, C, D, E, F groups of experimental rats were established the model of chronic high intraocular pressure (IOP) by cauterizing of superficial scleral vein.Animal model was established successfully by using monitoring IOP consistently keep above 25mmHg for 8wk as cut-off criterion.Tissues of Eyes were obtained after intragastric administration for 2wk and 4wk.The expressions of PAX6, Ngn1, and Ngn2 mRNA were investigated by Real-time PCR.RESULTS:At the time-point of 2wk, PAX6, Ngn1, and Ngn2 mRNA in group B were statistically expressed in lower level comparing with other groups (P0.05).CONCLUSION:In summar, Qingguang'an Ⅱ and Yimaikang disket can remarkably increase the expressions of PAX6, Ngn1, and Ngn2, which suggest protecting the optic nerve of rats caused by chronic high IOP.What's more, this study indicated that, in the protection of optic nerve of rats with chronic high IOP, the high dose of Qingguang'an Ⅱ at the time-point of 4wk was the better choice.
ABSTRACT
The purpose of the study was to investigate the impact of rat cytomegalovirus(RCMV) infection on the development of the nervous system in rat embryos,and to evaluate the involvement of Wnt signaling pathway key molecules and the downstream gene neurogenin 1(Ngn1) In RCMV infected neural stem cells(NSCs).Infection and control groups were established,each containing 20 pregnant Wistar rats.Rats in the infection group were inoculated with RCMV by intraperitoneal injection on the first day of pregnancy.Rat E20 embryos were taken to evaluate the teratogenic rate.NSCs were isolated from E13 embryos,and maintained in vitro.We found:1) Poor fetal development was found in the infection group with low survival and high malformation rates.2) The proliferation and differentiation of NSCs were affected.In the infection group,NSCs proliferated more slowly and had a lower neurosphere formation rate than the control.The differentiation ratio from NSCs to neurons and glial cells was significantly different from that of the control,showed by immunofluorescence staining.3) Ngn1 mRNA expression and the nuclear β-catenin protein level were significantly lower than the control on day 2 when NSCs differentiated.4) The Morris water maze test was performed on 4-week pups,and the infected rats were found worse in learning and memory ability.In a summary,RCMV infection caused abnormalities in the rat embryonic nervous system,significantly inhibited NSC proliferation and differentiation,and inhibited the expression of key molecules in the Wnt/β-catenin signaling pathway so as to affect NSCs differentiation.This may be an important mechanism by which RCMV causes embryonic nervous system abnormalities.
ABSTRACT
Objective To investigate the influence of murine cytomegalovirus ( MCMV) infection on the expression of downstream differentiation related target genes of Wnt signaling pathway in neural stem cells (NSCs) in vitro and explore the molecular mechanism of fetal encephalodysplasia caused by CMV infection. Methods NSCs were separated from fetal BALB/c mouse and cultured in vitro. The NSCs infected by MCMV at a MOI (multiplicity of infection) of 5, 1 and 0.1, respectively, were cultured in differentiation medium. The dynamic expression of the downstream differentiation related target genes ( c-myc, cyclinD1, ngn-1 and ngn-2) of Wnt signal pathway in NSCs were measured by Western blot. Real-time RT-PCR was employed to measure the expression levels of the key differentiation genes ngn-1 in Wnt signal pathway of NSCs post infection. Results The protein levels of c-myc in the infected groups were significantly lower than that in the normal control at 0.5-5 d (P＜0.05) ; At 0. 5 d and 1 d post-infection (p. i. ) , the protein levels of cyclinDl in the infected groups were lower than that in the normal control (P＜0.05). At 2 d and 3 d p. i. , the cyclinD1 expression in the infected groups was higher than that in the control group (P ＜ 0. 05). However, at 4 d and 5 d p. i. , the cyclinD1 levels in the group of the MOI of 5 were lower than in other three groups (F＜0.05). The expression of ngn-1 protein in the infected groups was reduced importantly compared with normal control at 1 -5 d p. i. ( P ＜ 0.05 ). The expression of ngn-1 mRNA in the infected groups was lower than that in the control group at all time points (P ＜ 0. 05 ). The expression of ngn-2 protein decreased at first and then increased, which was opposite to the normal control. The peak of ngn-2 expression in groups of the MOT of 0.1 and 1 occurred later and were significantly lower than that in the normal control (P ＜0. 05). No distinct peak was seen in the group of the MOI of 5. At 1 d p. i. , the expression of ngn-2 of all infected groups was significantly lower than that in the normal control ( P ＜ 0. 05 ). At 2 d p. i. , the expression of in the group of the MOI of 5 was still lower (P ＜ 0.05). While at 3 d, 4 d and 5 d p. i. , the protein levels in all infected groups were higher than that in the normal control (P ＜ 0. 05). The protein expression of these genes increased following the increase of MOI. Conclusion MCMV inhibited the protein expression of c-myc and ngn-1 in differentiated NSCs, repressed the mRNA expression of ngn-1 and caused the perturbed expression of cyclinDl and ngn-2 in a MOI-dependent manner. These data suggest that inhibition of or interference with the protein expression of downstream differentiation related target genes of Wnt signaling pathway in NSCs by MCMV may be one of the important mechanisms, by which proliferation and differentiation of NSCs are inhibited and thus fetal brain is impaired after MCMV infection.