Effect Of MCMV Infection On The Number Of Hippocampus Synapse And The Expression Of Related Proteins In Neonate Mice

Part 1 Effect of MCMV infection on the number of hippocampus synapse and the expression of related proteins in neonate miceObjective To study the effect of MCMV infection on the number of hippocampus synapse and the expression of related proteins in neonate mice.Methods①MCMV smith strain was duplicated by cell culture in vitro, and the virulence was measured with Reed-Muench method.②The model of MCMV infection on neonate mice was built as follows:the BALB/C mice with negative MCMV IgG and IgM were screened and mated. The fetal mice were randomly divided into experimental group(n=90) and control group(n=90) on the day of delivery. The fetal mice in experimental group were inoculated via intracalvarium with 10μl of MCMV suspension, the mice in control were inoculated with identity volume of cell maintenance medium. On the 3rd,15th,30th days post-inoculation, the brain tissues of 30 mice in each group were obtained asepticly. The mice with MCMV infection were detected by PCR amplification. The brain tissues of MCMV DNA positive in experimental group and MCMV DNA negative in control group were reserved for further research.③The numbers of hippocampus synapse in fetal mice were detected by immunofluorescence.④The transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor were detected with RT-PCR, immunohistochemistry and western blotting.Results①The TCID50of MCMV Smith strain was 10-4.5 per 0.1ml.②MCMV DNA in brain tissue was positive in experimental group, while that was negative in control group.③The fluorescence granules of SYP increased as days going on in both groups. But the fluorescence granules of SYP in experimental group were less than that in control, the decrease was more obvious on days 15th and 30th.④The transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor were increasing as days going on. But the transcription and protein expression of SYP PSD-95 and GluR2 subunit of AMPA receptor in experimental group were lower than that in control group, the decrease was more obvious on days 15th and 30th.Conclusions①MCMV infection could result in the decrease of hippocampus synapse in fetal mice, this effect increased as the days going on.②MCMV infection could induce the decrease of the transcription and protein expression of SYP, PSD-95 and GluR2 subunit of AMPA receptor in developing fetal mice. Objective To investigate the effect of secretory products of MCMV infected astrocytes on synapse quantity and expression of synapse-related protein.Methods①Establish MCMV infected astrocyte model:primarily culture and subculture neonatal rat hippocampal astrocytes; identification and purity of astrocytes were observed by using Immunohistochemistry to detect specific marker GFAP expression; Infect astrocytes with 100TCID50 MCMV, and then use the following methods to observe infection susceptibility and status of astrocyte to MCMV, growth situation of MCMV was also detected:PCR to detect MCMV DNA, CCK-8 to observe the survival rate of MCMV infected astrocytes, observing the cytotoxic effect of culture media on NIH 3T3 cell, drawing a growth curve of MCMV ect.②Establish the co-culture system of ACM(astrocyte conditioned medium) and neural cells in vitro:primarily culture neonatal rat hippocampal neurons; NSE(neuron specific enolase) expression and the purity of neurons were observed by Immunohistochemistry; collect ACM in the six-well plate after MCMV infection within 6h,12h,24h,48h,72h and that of control groups at the same time points, after filtration and centrifugation MCMV in the ACM was inactivated by ultraviolet light irradiating 15min; CCK-8 method was used to dentify the optimal concentration of ACM used in follow-up studies; the study is grouped according to various components of ACM:experiment group (including optimal concentration at different time points of infected ACM), ACM controls (including optimal concentration at different time points of noninfected ACM) and control group (without ACM).③Immunofluorescence was applied to detection the number of synapses.④Real-time qPCR and immunocytochemistry were used to detect the gene transcription and protein expression levels of synaptic associated protein SYP, PSD-95 and AMPA receptor GluR2 subunit.Results①Typical cytopathy occured after MCMV infection in cultured astrocytes (>95%purity); expression of MCMV IE DNA can be detected in infected cells at the 48h time point; ACM of 3d-4d after infection induced typical CMV virus plaque in NIH 3T3 cells; astrocytes began to die 24h after infecton, most died 72h after infection, at 96h time point there were no survivers; MCMV replication began to increase 12h after infection and peaked at 72h.②Purity of cultured neurons can be more than 98%, the neuron survival rate reach summit when medium containing 50%ACM.③Compared with the control group, ACM control and experiment groups, the number of 12h-72h SYP fluorescent particles significantly increased (P<0.01); with ACM control group, experimental group 12h-72h number of SYP fluorescent particles significantly reduced (P<0.01). Comparison group within-sample found, ACM control group 12h,24h,48h SYP fluorescent particles was significantly higher than the prior temporary phase (P<0.01),72h did not change significantly (P>0.05); experiment group only 12h the number of SYP fluorescent particles significantly increased than the prior temporary phase (P<0.01),24h,48h,72h did not change significantly (P>0.05).④ompared with the control group, ACM control and experiment groups 12-72h of three proteins' coding gene transcription and protein expression levels were significantly higher (P<0.01). Compared with ACM control group, the experiment group 12-72h of three proteins' coding gene transcription and protein expression levels were significantly lower (P<0.01). Comparison group within-sample found, ACM control group 12h,24h,48h three proteins' coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.01), 72h did not change significantly (P>0.05); experiment group, only 12h coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.0.1),24h,48h,72h did not change significantly (P> 0.05).Compared with the control group, ACM control and experiment groups 12-72h of coding gene transcription and protein expression levels were significantly higher (P<0.01). Compared with ACM control group, the experiment group 12-72h of coding gene transcription and protein expression levels were significantly lower (P0.05); experiment group, only 12h coding genes transcription and protein expression levels were significantly higher than the prior temporary phase (P<0.0.1),24h,48h,72h did not change significantly (P> 0.05).Conclusion①MCMV can replicate and induce cytopathic effects in astrocyte.②Astrocyte secretion can promote the formation of synapse, and MCMV may reduce the formation by making an effect on the synthesis and secretion function of astrocyte.③Astrocyte secretion can promote the coding gene transcription and protein expression of synaptic associated proteins, MCMV may influence the gene transcription and protein expression by disturb the secretory function of astrocyte. Objective To study the effect of MCMV infection on TSP-1 and TNF-a synthesized and excreted by astrocyte.Methods The astrocyte cells were cultured and then divided into experimental and control groups.100 TCID50MCMV and cell maintenance medium were added into the medium of experimental group, while in control, only cell maintenance medium was added. After 6,12, 24,48 and 72h, the cultured cells and astrocyte conditioned medium (ACM) were collected respectively. The ACM was exposed to ultraviolet rays for 15 minutes to inactivate CMV, after filtration and centrifugalization.①The mRNA and protein expression of TSP-1 and TNF-a were detected by RT-PCR, immunohistochemistry.②TSP-1 and TNF-a excreted by astrocyte were detected with ELISA method.Results①Comparing to control group, the mRNA and protein expression of TSP-1 and TNF-a were lower in experimental group at each time point. The diversity increased as the time going on. Furtherly, the mRNA and protein expression of TSP-1 and TNF-a decreased during 12h-72h post-culturing.②By ELISA, TSP-1 and TNF-a in ACM can be detected from 12h post-culturing. TSP-1 and TNF-a in control showed a sharp increasing during 0-48h, and then increased mildly during 48-72h. While in experimental group, they were invariable during 12-72h. At each time point, TSP-1 and TNF-a in experimental group were lower than that in control.Conclusions①TSP-1 and TNF-a synthesized and excreted by astrocyte decreased after CMV infection. As the time going on, the decrease was more distinguished.②TSP-1 and TNF-a excreted by astrocyte increased in normal, while it was inhibited by CMV infection. The excretion of TSP-1 and TNF-a maintained in low level with CMV infection.