体外共培养潜伏感染的老年小鼠大脑皮质及肺组织内HCMV研究
发布时间：2019-04-19 22:51
【摘要】：目的 取HCMV先天性潜伏感染小鼠脑、肺组织并制成细胞悬液,体外与HF细胞进行共培养,观察细胞中潜伏的病毒再激活现象,为研究HCMV潜伏再激活及阐明机制提供证据和新线索。 方法 依据随机抽样原则,取HCMV先天性潜伏感染组小鼠及DMEM对照组小鼠各6只,分别将其大脑皮质和肺组织制成细胞悬液。染色计数活细胞,细胞终浓度为1×10~6/ml,接种至放有盖玻片的细胞培养板中。随即加入1×10~6/ml的人胚成纤维细胞(HF)悬液,建立共培养体系。同时设立HCMV阳性对照和HF阴性对照。逐同观察细胞生长情况并记录。分别在共培养后的第1、3、7、14、28、35d收获细胞,进行如下检测:(1)观察共培养物上HCMV特征性CPE;(2)PCR、RT-PCR分别检测HCMV IE、UL83 DNA和相应的mRNA;(3)取共培养细胞爬片做间接免疫荧光试验,分别检测HCMV IE和pp65抗原的表达;(4)透射电镜观察疱疹病毒样颗粒和感染的HF细胞超微结构变化。 结果 HCMV先天性潜伏感染的老年小鼠大脑皮质和肺组织细胞体外分别与HF共培养组:(1)HCMV潜伏感染的老年小鼠大脑皮质和肺组织细胞经体外与HF共培养,于第7d开始出现HCMV特征性CPE,病变呈灶性并逐渐扩展至整个单层,病变特征同病毒阳性对照。潜伏病毒再激活率为100%。(2)PCR扩增出HCMVIE、UL83 DNA且序列测定与Genbank上登录的基因序列比较,一致率分别为99%和100%。RT-PCR检测见HCMVIE、UL83特异性条带,HCMV mRNA在第3d开始表达。HCMV基因的复制具有明显的时序性,HCMV IE基因的表达及转录均早于UL83,随着时间的延长,表达水平逐渐升高。(3)间接免疫荧光试验于共培养后第7d检出IE、pp65蛋白。(4)透射电镜下观察到HF细胞超微结构明显病变,发现典型的疱疹病毒样颗粒。DMEM对照组及HF阴性对照组均末检测到所设置
[Abstract]:Objective to obtain the brain and lung tissue of HCMV congenital latent infection mice and make cell suspension, and to co-culture with HF cells in vitro to observe the reactivation of latent virus in the cells. It provides evidence and new clues for studying the mechanism of latent reactivation and elucidation of HCMV. Methods according to the principle of random sampling, 6 mice in HCMV congenital latent infection group and 6 mice in DMEM control group were used to make cell suspension from cerebral cortex and lung tissue respectively. The cells were stained and counted, and the final cell concentration was 1 脳 10? 6? ml. The cells were inoculated into the cell culture plate with cover glass. The co-culture system was established by adding 1 脳 10 ~ 6/ml human embryonic fibroblast (HF) suspension. At the same time, HCMV positive control and HF negative control were established. Cell growth was observed and recorded. The cells were harvested at 1,3,7,14,28,35 days after co-culture, respectively. The results were as follows: (1) the characteristic CPE; (2) PCR,RT-PCR of HCMV on the co-culture medium was observed to detect HCMV IE,UL83 DNA and corresponding mRNA;, respectively. (3) the expression of HCMV IE and pp65 antigen were detected by indirect immunofluorescence assay, and (4) the ultrastructural changes of herpes virus-like granules and infected HF cells were observed by transmission electron microscopy. Results the cells of cerebral cortex and lung of aged mice with congenital latent infection of HCMV were co-cultured with HF in vitro. (1) the cells of cerebral cortex and lung of aged mice infected with HCMV latent infection were co-cultured with HF in vitro. On the 7th day, the characteristic CPE, lesions of HCMV appeared focal and gradually extended to the whole monolayer, and the pathological features of the lesions were compared with those of the virus positive controls. The reactivation rate of latent virus was 100%. (2) HCMVIE,UL83 DNA was amplified by PCR and compared with the gene sequence logged on Genbank, the coincidence rate was 99% and the specific band of HCMVIE,UL83 was detected by 100%.RT-PCR, respectively. HCMV mRNA began to express on the 3rd day. The replication of HCMV gene had obvious time sequence, HCMV IE gene expression and transcription earlier than that of UL83, with the prolongation of time. (3) IE,pp65 protein was detected by indirect immunofluorescence assay on the 7th day after co-culture. (4) obvious ultrastructural changes of HF cells were observed under transmission electron microscope (TEM). Typical herpesvirus-like granules were found in both the DMEM control group and the HF-negative control group.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
本文编号：2461362
[Abstract]:Objective to obtain the brain and lung tissue of HCMV congenital latent infection mice and make cell suspension, and to co-culture with HF cells in vitro to observe the reactivation of latent virus in the cells. It provides evidence and new clues for studying the mechanism of latent reactivation and elucidation of HCMV. Methods according to the principle of random sampling, 6 mice in HCMV congenital latent infection group and 6 mice in DMEM control group were used to make cell suspension from cerebral cortex and lung tissue respectively. The cells were stained and counted, and the final cell concentration was 1 脳 10? 6? ml. The cells were inoculated into the cell culture plate with cover glass. The co-culture system was established by adding 1 脳 10 ~ 6/ml human embryonic fibroblast (HF) suspension. At the same time, HCMV positive control and HF negative control were established. Cell growth was observed and recorded. The cells were harvested at 1,3,7,14,28,35 days after co-culture, respectively. The results were as follows: (1) the characteristic CPE; (2) PCR,RT-PCR of HCMV on the co-culture medium was observed to detect HCMV IE,UL83 DNA and corresponding mRNA;, respectively. (3) the expression of HCMV IE and pp65 antigen were detected by indirect immunofluorescence assay, and (4) the ultrastructural changes of herpes virus-like granules and infected HF cells were observed by transmission electron microscopy. Results the cells of cerebral cortex and lung of aged mice with congenital latent infection of HCMV were co-cultured with HF in vitro. (1) the cells of cerebral cortex and lung of aged mice infected with HCMV latent infection were co-cultured with HF in vitro. On the 7th day, the characteristic CPE, lesions of HCMV appeared focal and gradually extended to the whole monolayer, and the pathological features of the lesions were compared with those of the virus positive controls. The reactivation rate of latent virus was 100%. (2) HCMVIE,UL83 DNA was amplified by PCR and compared with the gene sequence logged on Genbank, the coincidence rate was 99% and the specific band of HCMVIE,UL83 was detected by 100%.RT-PCR, respectively. HCMV mRNA began to express on the 3rd day. The replication of HCMV gene had obvious time sequence, HCMV IE gene expression and transcription earlier than that of UL83, with the prolongation of time. (3) IE,pp65 protein was detected by indirect immunofluorescence assay on the 7th day after co-culture. (4) obvious ultrastructural changes of HF cells were observed under transmission electron microscope (TEM). Typical herpesvirus-like granules were found in both the DMEM control group and the HF-negative control group.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
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