| Objective:1. To detect the expression of human cytomegalovirus genes and proteins in infected human embryo fibroblast (HEF) and mouse primary embryo fibroblast (MEF) cells for the further research of HCMV species-specificity mechanisms.2. To investigate the different effects of HCMV on autophagy in MEF and HEF cells.Methods:1. HEF and MEF primary cells were isolated and cultured by the method of tissue piece combining with collagenase digestion and pured by anchorage velocity-dependent separation method.2. HEF cells were infected with HCMV to amplify the virus; Plaque assay was applied to quantitative determination of virus titer.3. The morphological changes were observed by phase contrast microscope after infection (MOI=5).4. Expression of HCMV Immediate-early gene (IE1, IE2), early gene (UL84) and late gene (UL83) in HCMV-infected MEF and HEF cells was detected by RT-PCR; The related protein was detected by Western blot and immunofluorescence.5. The proliferation of the both infected cells was measured by MTT.6. Autopagosomes in HEF and MEF cells were observed by electron microscopy after monodansylcadaverine (MDC) staining.7. The expressions of Beclinl and LC3in both cells were detected by Western blotting and LC3was also observed by immunofluorescence.Results:1. HEF and MEF primary cells were isolated and cultured successfully in vitro.2. Determination results of plaque assay showed the average plaques number was7when HCMV was diluted10-4times and added5ul. The calculation confirms that the plaque forming unit (PFU) was1.4×107PFU/ml.3. Observations through phase contrast microscope showed that CMV-specific cytopathic effects (CPE) appeared at day4post inculation in more than80%HCMV AD169-infected HEF cells but MEF did not display the significant HCMV-specific cytopathic effect (CPE) like that.4. RT-PCR and western blot results showed the infected MEF could express IEI, IE2and corresponding proteins, which was apparently higher than negative control (P<0.01), whereas IE1, IE2, UL84, UL83genes and proteins were expressed in HEF, which was obviously lower than HEF group (P<0.05), but higher than non-infected control (P<0.01); Immunofluorescence results showed proteins coded by IE and UL83genes were found obviously in HEF cells, but not in MEF cells at72h.5. The proliferation index of the both infected cells indicated by MTT was increased and had no significantly difference at48h. But the proliferation of the HEF cells was inhibited significantly at72h while that of the MEF cells was not down.6. Autopagosomes were found in the both cells at6h under fluorescence microscope by MDC staning and election microscopeincreased. The number of autopagosomes increased at6h after infection in both groups, but reached to the highest level at24h, and then began to decline in HEF cells, which were still higher than control group. While the MEF cells still increased.7. Western blotting and immunofluorescence results showed the expression of Beclinl and LC3increased at6h after infection in both cells, but reached to the highest level at24h, and then began to decline in HEF cells, which were still higher than that in control group. However, the MEF cells still showed an gradual increased trend.Conclusions:1. HCMV could not cross the species-specificity to infect MEF and HCMV gene expression in MEF was restricted after IE2and before UL84expression, and the species-specificity mechanisms of HCMV perhaps were related with the less expressed IE genes.2. In the infected HEF, autophagy was inhibited after24h, by contrast, HCMV enhances autophagy levels in MEF cells3. There may be some relationship between different effects of HCMV on autophagy in human and mouse primary embryonic cells and HCMV genes expression in both cells... |
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