感染伪狂犬病病毒对小鼠脑细胞周期及相关因子的影响

Effects of Pseudorabies virus infection on brain cell cycle-related factors in mice

为了研究感染猪伪狂犬病病毒(Pseudorabies virus,PRV)对小鼠脑细胞周期及相关因子的影响,试验将40只6周龄Balb/c小鼠随机分成对照组和36小时组、48小时组、60小时组,试验组经皮下接种含量为1×10^(3)TCID_(50)/100μL的PRV溶液200μL,对照组注射等量生理盐水。试验采用流式细胞术检测脑细胞周期变化,并采用实时荧光定量PCR和Western-blot方法分别检测细胞周期相关因子基因和蛋白质的表达情况。结果表明:与对照组相比,感染PRV后36小时,处于G0/G1期的细胞比例极显著升高,而处于S期的细胞比例则极显著降低(P<0.01)。感染PRV后36小时,CDK4和CDK6基因相对表达量显著和极显著下降(P<0.05,P<0.01),60小时时恢复到对照组水平(P>0.05);感染PRV后36,48小时,cyclin D基因相对表达量极显著降低(P<0.01);而在感染PRV后36小时时cyclin E基因相对表达量显著上升(P<0.05),60小时时极显著上升(P<0.01)。此外,感染PRV后36小时,CDK4和CDK6蛋白相对表达量出现了下调;感染PRV后36,48小时,cyclin D蛋白处于下调表达,在感染后60小时略有上调;而在整个观察期内,cyclin E蛋白一直处于上调表达。说明感染PRV的早期,宿主可通过调节多种细胞因子间相互作用将细胞周期阻滞在G0/G1期。

In order to investigate the effects of porcine Pseudorabies virus(PRV)infection on the brain cell cycle and related factors in mice,406-week-old Balb/c mice were randomly divided into control group and 36h,48h,and 60h groups.The test group was inoculated subcutaneously with 200μL of PRV solution at a level of 1×10^(3)TCID_(50)/100μL,and the control group was injected with an equal amount of normal saline.The experiment used flow cytometry to detect brain cell cycle changes,and real-time fluorescence quantitative PCR and Western-blot to detect the expression of cell cycle-related factor genes and proteins,respectively.The results showed that compared to the control group,at 36 h after PRV infection,the proportion of cells in the G0/G1 phase was highly significantly higher and the proportion of cells in the S phase was highly significantly lower(P<0.01).At 36 h after PRV infection,the relative expression of CDK4 and CDK6 genes significantly and very significantly decreased(P<0.05,P<0.01),and returned to control group levels at 60 h(P>0.05).At 36,48 h after PRV infection,the relative expression of cyclin D gene was highly significantly reduced(P<0.01).While at 36 h after PRV infection,the relative expression of cyclin E gene increased significantly(P<0.05)and highly significantly increased at 60 h(P<0.01).In addition,At 36 h after PRV infection,the relative expression of CDK4 and CDK6 proteins was down-regulated;at 36 and 48 h after PRV infection,cyclin D protein was in down-regulated expression and was slightly up-regulated at 60 h post infection;while throughout the observation period,cyclin E protein was in up-regulated expression.This study suggested that at the early stage of PRV infection,the host could block the cell cycle in G0/G1 phase by regulating the interaction among various cytokines.