人巨细胞病毒感染小鼠大脑皮质细胞的体内外实验研究

发布时间：2018-05-27 20:00

本文选题：人巨细胞病毒 + 小鼠大脑皮质细胞　；参考：《安徽医科大学》2006年硕士论文

【摘要】：目的：研究人巨细胞病毒(human cytomegalovirus，HCMV)在体外可以增殖性感染新生小鼠大脑皮质细胞；在体内可以先天性潜伏感染小鼠大脑皮质细胞，并且可以被再激活。方法：体外试验：选用HCMV AD169株和3株HCMV临床分离株作为本次实验的毒种，并设立经高温灭活后HCMV AD169株对照组。对所有毒株进行50％组织细胞感染量(TCID_(50))测定，然后在长成单层的Balb／c新生小鼠的大脑皮质细胞和HF细胞6孔板上分别接种相同滴度的病毒悬液0.1ml／孔。逐日镜下观察HCMV特征性细胞致病变效应(CPE)，同时采用PCR、RT-PCR、间接免疫荧光检测HCMV特异性基因IE和UL83的复制和蛋白表达，确定HCMV感染状态。分别在感染后第1、2、3、4、5天收集细胞及其培养上清液，采用TCID_(50)法检测被感染的新生小鼠大脑皮质细胞培养物上清的病毒滴度，每种病毒标本分两组，每组重复三次；并且在不同的时间点固定细胞片进行HCMV特异性早期蛋白和pp65蛋白免疫荧光检测；透射电镜观察大脑皮质细胞内HCMV病毒颗粒。体内试验：利用已建立的HCMV先天性潜伏感染的小鼠模型。试验分为三组：HCMV先天潜伏感染组、HCMV先天性潜伏感染再激活组(小鼠腹腔注射环磷酰胺150mg／kg，每6天1次，共3次)、HF细胞对照组小鼠。在各组小鼠达18～20月龄时，，分别在各组随机选取小鼠5只。取HCMV先天性潜伏感染组、HCMV先天性潜伏感染再激活组小鼠和HF细胞对照组小鼠大脑皮质细胞与HF细胞建立共培养体系。利用PCR、RT-PCR、间接免疫荧光检测HCMV特异性基因IE和UL83的复制和蛋白表达。结果：体外试验研究：将TCID_(50)为1.7×10~3／ml HCMV AD169株病毒悬液接种在新生小鼠大脑皮质细胞培养物上，在感染后第2天可见神经细胞形态明显改变：镜下
[Abstract]:Aim: to investigate whether human cytomegalovirus (HCMV) can proliferate and infect neonatal mouse cerebral cortex cells in vitro and can be reactivated in vivo by congenital latent infection. Methods: in vitro test: HCMV AD169 strain and 3 clinical isolates of HCMV were selected as the virus strains in this experiment, and the control group of HCMV AD169 strain after high temperature inactivation was set up. All the strains were detected by 50% histocyte infection and then inoculated with the same titer of virus suspension 0.1ml/ holes on the 6-well plates of the cerebral cortex cells and HF cells of the monolayer Balb/c newborn mice. The characteristic cytopathic effect of HCMV was observed daily. Meanwhile, the replication and protein expression of HCMV specific genes IE and UL83 were detected by indirect immunofluorescence, and the status of HCMV infection was determined. The cells and their supernatants were collected on the first day after infection and the supernatant of culture was collected. The virus titers of the supernatant of cerebral cortex cell culture of infected newborn mice were detected by TCID-50 method. Each kind of virus specimen was divided into two groups and repeated three times in each group. The HCMV specific early protein and pp65 protein were detected by immunofluorescence at different time points, and the HCMV virus granules in cerebral cortex cells were observed by transmission electron microscope. In vivo test: a mouse model of congenital latent infection of HCMV was established. The experiment was divided into three groups: HCMV congenital latent infection group and HCMV congenital latent infection reactivation group (mice were injected cyclophosphamide 150 mg / kg, once every 6 days, 3 times). At the age of 18 to 20 months, 5 mice were randomly selected in each group. The coculture system of cerebral cortex cells and HF cells was established in mice of HCMV congenital latent infection group and HF cell control group. The replication and protein expression of HCMV specific genes IE and UL83 were detected by indirect immunofluorescence. Results: in vitro, the virus suspension of 1. 7 脳 10~3/ml HCMV AD169 strain was inoculated on the culture of cerebral cortex cells of newborn mice. The morphology of neurons was obviously changed on the second day after infection.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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