HCMV感染对HEL细胞MCM装载因子Cdt1的影响

Influence of HCMV infection on MCM-loading factor Cdt1 in HEL cells

目的研究人类巨细胞病毒(HCMV)感染对人胚肺成纤维(HEL)细胞的MCM装载因子Cdt1表达的影响,从分子水平上探讨HCMV的发病机制。方法用血清饥饿法同步化HEL细胞于G0/G1期,用HCMV感染同步化的HEL细胞作为感染组,同时设模拟感染组为对照组。于感染后12、24、48、72和96h收获细胞,用免疫细胞化学法检测两组细胞核内HCMVCdt1蛋白。结果 12、24、和96h两组HEL细胞Cdt1蛋白水平均差异有显著性(P<0.05),而48、72h两组HEL细胞Cdt1蛋白水平无明显差异(P>0.05)。12和96h感染组较模拟感染组明显降低,24h感染组较模拟感染组明显升高。模拟感染组在感染后12h时Cdt1蛋白表达水平最高,然后逐渐下降,至24h时达最低水平,96h又稍有回升;感染组在感染后12hCdt1蛋白表达水平较低,24h达到最高峰,48h时急剧下降,一直维持较低的稳定水平。结论 HCMV诱导Cdt1蛋白表达延迟,从而影响了MCM复合物的装载和pre-RC的形成,使细胞停滞在G1期,阻止DNA的复制。

[Objective] To investigate the influence of HCMV infection on MCM-loading factor Cdt1 in human embryonic lung fibroblastic （HEL） cells and the pathogenesis of HCMV infection from molecular level.[Methods] HEL cells in G0/G1 were synchronized by serum starvation. Synchronized HEL cells were infected with HCMV, at the same time the mock-infected group was set up as control group. Infected HEL cells were harvested at 12 hours post infection （h p. i.）, 24 h p.i., 48 h p.i., 72 h p.i. and 96 h p.i., Cdt1 protein was detected by immunocytochemistry method.[Results]There were significant difference in expression levels of Cdt1 protein in two groups （P 0.05） at 12 h p.i., 24 h p.i., 96 h p.i., but not at 48 h p.i., 72 h p.i （P 0.05）. Cdt1 protein in HCMV-infected group were statistically lower than the ones in the mock-infected group at 12 h p.i. and 96 h p.i , but at 24 h p.i. the expression levels of Cdt1 protein in HCMV-infected group were statistically higher than the ones in the mock-infected group. The expression levels of Cdt1 protein reached at a peak at 12 h p.i., reached a nadir at 24 h p.i., but then returned a little at 96 h p.i. in the mock-infected group; while the expression levels of Cdt1 protein were low at 12 h p.i., reached a peak at 24 h p.i., and then decreased sharply at 48 h p.i, maintained constantly lower stable level in HCMV-infected. [Conclusion]HCMV can induce the expressions delaying of Cdt1, lead to affect the loading of MCM compounds and the formation of pre-RC. Cell cycle is arrested at G1 phase, replication of DNA is forbidden.