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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: We established a mouse model of latent cytomegalovirus activation in mice to activate the latent cytomegalovirus (MCMV) in the mouse brain and analyzed the time course of latent MCMV activation to determine the MCMV immediate early protein gene 1 ( ie1) gene transcription, to complete the ie1 gene transcription and live virus generation kinetic analysis, as well as to further clarify the original MCMV latent cell types in the brain to provide a model. Methods: Two-day-old BALB / c pups were injected with Smith Strain MCMV 500 PFU / 5 μL into the right ventricle via the right triangle and the center of the equilateral triangle with the bottom of the eye for 16 weeks. Afterwards, lipopolysaccharide (LPS) was intraperitoneally and intracerebroventricularly injected according to 15μg / kg body weight (approximate lethal dose), and the control group was injected with normal saline. Five mice were selected from the LPS group and the control group on the 1st, 2nd, 5th, 7th, 14th and 21st days after injection, respectively. The expression of MCMV immediate early protein 1 (IE1) mRNA and the production of live virus were determined by reverse transcription-polymerase chain reaction (RT-PCR) and hypersensitive virus plaque assay (combined with viral plaque assay and RT-PCR) Quantitative analysis. Results: In the LPS group, the transcription of IE1 mRNA was detectable in the brain on the 14th and 21st days. The virus virulence (CPE) was only detected on the 14th and 21st days in the sensitive virus plaque experiment. The viral load was about 4.29 × 104 PFU / μL and 5.20 × 105 PFU / μL respectively. The results of RT-PCR of corresponding MEF cell homogenates showed that IE1 mRNA was transcribed on the 7th, 14th and 21st days. CONCLUSION: This experiment successfully established a model of mouse brain latent cytomegalovirus activation and confirmed and analyzed the immediate early protein gene ie1 expression and time course in latent MCMV activation. The establishment of this model will provide a research platform for further clarifying the types of latent MCMV cells in the brain and the influence of MCMV on CNS during acute infection, latent and reactivation, and provide an experimental study on the clinical research of human cytomegalovirus (HCMV) in accordance with.