小鼠脑潜伏巨细胞病毒激活模型的建立  

An Activation Model of Latent Murine Cytomegalovirus

作　　者：王宇[1] 刘军[1] 刘营营[1] 韩桂萍[1] 

机构地区：[1]哈尔滨医科大学附属第二医院病理科,黑龙江哈尔滨150086

出　　处：《现代生物医学进展》2015年第23期4414-4418,共5页Progress in Modern Biomedicine

基　　金：黑龙江省科技厅留学资金项目(LC08C17);哈尔滨市科技局科技创新人才研究专项资金(2008RFLXS012)

摘　　要：目的:我们建立了小鼠脑潜伏巨细胞病毒激活模型,来实现小鼠脑内潜伏的巨细胞病毒(MCMV)的激活,并对潜伏MCMV激活时程进行分析,确定MCMV即刻早期蛋白基因1(ie1)基因转录,完成对ie1基因转录和活病毒产生量时程动力学的分析,以及为进一步阐明原始MCMV在脑中潜伏的细胞类型提供模型。方法:采用出生后两天的BALB/c幼鼠,经右侧耳和眼连线为底边的正三角形的中心将Smith Strain MCMV 500 PFU/5μL注射进入右侧脑室,培养至16周。之后,将脂多糖(LPS)依15μg/kg体重(接近致死量)分别经腹腔和侧脑室内注射,对照组注射生理盐水。于注射后的1日,2日,5日,7日,14日和21日分别在LPS组和对照组中选取5只小鼠取脑。应用反转录-聚合酶链式反应(RT-PCR)和高敏感性病毒空斑实验(结合病毒空斑实验和RT-PCR)测定MCMV即刻早期蛋白1(IE1)mRNA的表达以及活病毒产生定量分析。结果:LPS组中,可于14日和21日的脑内检测到IE1 mRNA的转录,敏感性病毒空斑实验只在14日和21日出现细胞病毒效应(CPE),病毒量约为4.29×104 PFU/μL和5.20×105PFU/μL,相应对应MEF细胞匀浆物的RT-PCR结果检测到7,14,21日有IE1 mRNA转录。结论:该实验成功建立了小鼠脑潜伏巨细胞病毒激活的模型,并证实和分析了即刻早期蛋白基因ie1在潜伏MCMV激活过程中的表达和时程。该模型的建立将为进一步阐明MCMV在脑中潜伏细胞类型以及MCMV在急性感染、潜伏和重激活过程中对中枢神经细胞的影响提供研究平台,并为人巨细胞病毒(HCMV)的临床研究提供实验依据。Objective： To establish the activation of latent murine cytomegalovirus and analyze the schedule of activation; To confirm the transcription of MCMV immediate early protein 1（ie1） gene and complete the kinetics of transcription of ie1 gene and production of virus; To set up an activation model of latent MCMV for further research of the types of the cells where MCMV chromosome maintains during the period of latency. Methods： 2 days after birth, 500 PFU/5 μL of MCMV of minimum essential medium was injected into the right cerebral hemisphere of neonatal BALB/c mice. Control mice were similarly injected with minimum essential medium for 16 weeks. Intracranial injection and intraperitoneal injection of 15 μg/kg weight lipopolysaccharide（LPS） were administered, and saline was injected as a contrast. At 1, 2, 5, 7, 14 and 21 days postinfection, five mice from two groups were killed and the brains were removed. The brains were used for reverse transcriptase-polymerase chain reaction（RT-PCR） assays and sensitive plaque assays（combined plaque with RT-PCR） to detect the IE1 mRNA and virus titer. Results： IE1 mRNA was detected at 14 and 21 days in the group LPS. Cytopathic effect（CPE）was detected at 14 and 21 days, and titer were approximately 4.29×104 PFU/μL and 5.20×105PFU/μL. Transcription of IE1 mRNA was detected at 7, 14 and 21 days in the homogenate of mouse embryonic fibroblasts（MEFs）by sensitive plaque assays. Conclusions： The activation model of latent MCMV was established, ie1 mRNA was detected and the schedule of it during the period of activation. The model would be provided in further research of MCMV latency and activation in brains. It could also provide basis for the research of human cytomegalovirus（HCMV）.

关 键 词：小鼠巨细胞病毒 潜伏 激活 

分 类 号：Q953[生物学—动物学] R373[医药卫生—病原生物学]