Influence Of Human Cytomegalovirus Infection On Expressions Of Cell-cycle Associated Factor Cdt1 And Geminin Genes In HEL Cells

Objective To determine the expressions of cell-cycle associated factorCdt1 and Geminin genes in human embryonic lung fibroblastic(HEL)cells andthe influence of human cytomegalovirus(HCMV) infection on the expressions ofthese two genes, explore the pathogenesis of HCMV infection.Method The HEL cells were synchronized in G₀/G₁ phase by serumstarvation. Cell cycle of HEL cells was detected by flow cytometry analysis.Synchronized HEL cells were infected with HCMV, at the same time themock-infected group was set up as control group. During the time, observationof the pathomorphological change of infected HEL cells was performed until168 hours post infection by inverted phase contrast microscope. Infected HELcells were harvested at 12 hours post infection (h p.i.),24h p.i.,48h p.i.,72h p.i.and 96h p.i..The expressions of Cdt1 and Geminin genes were detected withsemi-quantitative RT-PCR method.Results1.The change of morphology was not observed in the mock-infected cells.In HCMV-infected group, the partial cells became round, bulgy, and theircytoplasm and nucleolus became large at 36h p.i., cytopathic effect(CPE) was observed in all infected cells at 168h p.i..2. The HEL cells cultured in DMCM respectively containing 0.5%, 1%,2% and 10% fetal bovine serum (FBS) for 48 hours showed different blockagepercentage at the G₀/G₁ phase, which were 82.8%, 78.4%, 69.9% and 43.8%.The HEL cells can be better synchronized in the FBS concentration of 0.5%, thesynchronized cell can be used for the further experiment.3. The statistical analysis showed the expression levels of Cdt1 gene inHCMV-infected group were statistically lower (P＜0.05)than the ones in themock-infected group at 12h p.i., 24h p.i., but at 48h p.i. and 72hp.i.,theexpression levels of Cdt1 gene in HCMV-infected group were statisticallyhigher(P＜0.05) than the ones in the mock-infected group. The graphs suggestedthat the expression levels of Cdt1 gene reached at a peak at 12h p.i. in themock-infected group;while at 48h p.i. in HCMV-infected group.4. The statistical analysis showed the expression levels of Geminin gene inHCMV-infected group were statistically higher (P＜0.05)than the ones in themock-infected group at 12h p.i., 24h p.i., but at 48h p.i. and 72hp.i.,theexpression levels of Geminin gene in HCMV-infected group were statisticallylower (P＜0.05) than the ones in the mock-infected group. The graphs suggestedthat the expression levels of Geminin gene reached at a peak at 12h p.i., in theHCMV-infected group;while at 48h p.i. in mock-infected.Conclusion1.Most HEL cells can be synchronized in G₀/G₁ by Serum starvation, and the DMCM with 0.5% FBS could get the best effect in synchronizing cells.2. HCMV can induce the abnormal expressions of Cdtl and Geminin genes,resulting in the geminin/Cdt1 imbalance, which could be responsible for theblockage in cell cycle in G₁ phase by HCMV infection.