人巨细胞病毒感染对宿主细胞周期进程影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞DNA合成及细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0／G1期的人胚肺成纤维细胞（HEL），分别于感染后0、3．6、12、24、48、72、96h终止培养。用流式细胞术测定HCMV感染后细胞周期进程及DNA含量。用免疫蛋白印迹法（Western Blot）检测CyclinE、Cyclin A、Cyclin D1蛋白的表达。结果HCMV感染细胞后24h-96h，S期细胞明显增多，G2／M期细胞减少，至感染后96h，未发现有G2／M期细胞。感染后24h细胞保持2N DNA含量，全部感染细胞DNA含量在48h内开始升高，感染后72h多数细胞的DNA含量大于2N DNA含量。在正常对照细胞DNA含量没有增加。HCMV＋PAA组没有检测到DNA含量增加。HCMV感染接触抑制细胞12h时CyclinE蛋白被诱导，感染后24h出现Cyclin E峰值；HCMV不能诱导Cyclin A蛋白表达；CyclinD1在感染后24h开始下降。结论HCMV感染同步化于G0／G1期的细胞后，诱导CyclinE蛋白明显升高，激活CyclinE／Cdk2激酶，使细胞周期越过G1／S限制点，将细胞周期阻止于晚G1期。病毒感染未能激活细胞DNA合成，病毒感染后总DNA的增加由病毒DNA复制造成。

Experimental study of the effect of human cytomegalovirus infection on cell cyqcle progression and the expression of cyclins

OBJECTIVE: To observe the effect of human cytomegalovirus infection on the host cellular DNA synthesis and expression of cyclones. METHODS: HCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres. The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell. We harvested infected cell at different time 0 h, 3 h, 6 h, 24 h, 72 h and 96 h post infection. Then the cell cycle progress was measured. Meanwhile, the DNA content and expression of proteins of cycline E, cycline A and cycline D1 were determined with FCM and Western Blot respectively. RESULTS: We found that the amount of S stage cell infected by HCMV had increased dramatically, and that of G2/M stage cell reduced during 24 h-96 h PI, and no G2/M stage cell was detected within 96 h PI. The content of 2N DNA maintained unchangeable for 24 h after infection and the content of total DNA in infected cells began to increase within 48 h PI, and the substantial cell with 2N DNA were observed 72 h after infection. However, DNA content was not altered in control group of normal HEL and HCMV PAA group. CyclinE protein was induced 12 h PI and peak induction occurred 24 h PI in contact-inhibited cells. CyclinA protein expression was not induced in HCMV infected density-arrested cells. The abundance of cyclinD1 decreased 24 h PI. CONCLUSION: The expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV, which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage. HCMV can not activate cellular DNA synthesis, and increase of total DNA content in infected cells result from the viral DNA replication.