人巨细胞病毒体外感染胎鼠大脑皮质细胞及其分子机制探讨

发布时间：2018-04-19 23:42

本文选题：人巨细胞病毒 + 增殖性感染　；参考：《安徽医科大学》2007年硕士论文

【摘要】： 目的研究人巨细胞病毒(human cytomegalovirus，HCMV)体外增殖性感染胎鼠大脑皮质细胞，初步探讨HCMV跨种属感染的分子机制。为临床研究HCMV中枢神经系统感染提供实验依据。方法将本实验室纯化后的HCMV AD169株通过空斑形成试验(plaque forming unit，PFU)测定病毒感染性。以4×10~5 PFU／ml病毒感染量接种至Balb／c胎鼠的大脑皮质细胞和人胚肺成纤维细胞(human embryo lung cell，HEL)，在感染后的第1、3、5和7天分别收集细胞培养物及其上清，用不同的方法检测如下指标：(1)HCMV接种后，通过倒置显微镜逐日观察HCMV致胎鼠大脑皮质细胞病变效应(cytopathic effect，CPE)，并观察其病变特征；(2)通过PCR和RT-PCR方法检测胎鼠大脑皮质细胞中与HCMV复制增殖的重要基因IE、UL83和UL54 DNA和mRNA；同时，以β-actin为内参，研究其mRNA含量的变化特征及趋势；(3)通过空斑形成试验检测HCMV感染胎鼠大脑皮质细胞病变效应达到+++～++++时感染性病毒数量；(4)通过透射电镜观察HCMV感染后胎鼠大脑皮质细胞胞内的病毒颗粒及其超微结构的变化。结果(1)将HCMV AD169毒株以4×10~5 PFU／ml接种至胎鼠大脑皮质细胞2天后，出现病毒致细胞病变效应(CPE)，可见少数神经元和胶质细胞细胞肿胀、变圆，胞内颗粒增多，折光性增强等病变。在第3、5和7天时，细胞病变数量逐渐增加，病变程度逐步加重，表现为轴突断裂、细胞溶解甚至脱落等情况。而接种病毒的HEL则在第1天就出现CPE，且随着时间的延长，细胞病变数目增多，病变加重，，表现为细胞肿胀、变圆、折光性增强、脱落等情况。相对来说，HCMV对HEL更为敏感、CPE出现早、细胞病变程度重；(2)HCMV接种至胎鼠大脑皮质细胞的第1、3、5和7天，PCR检测到细胞培养物HCMV IE、UL83和UL54 DNA均呈阳性；同时，以β-actin为内参，RT—PCR检测各时间点mRNA，结果表明：各时间点均可检测到HCMV IE、UL83和UL54 mRNA，产物大小分别为872bp、842bp和784bp，经Bioscience image anlysis system进行灰度值分析显示，随着HCMV感染胎鼠大脑皮质细胞时间的延长，HCMV IE、UL83和UL54 mRNA表达量均明显增加，证实HCMV在胎鼠大脑皮质细胞内为增殖性感染；(3)采用空斑形成实验结果表明：约在第7天，接种病毒悬液的HEL细胞层出现圆形空斑，随着感染天数的增加，空斑变大，数目增多。约14天空斑基本不在发生变化，根据公式计算得到病毒感染量为3.8×10~5PFU／ml。(4)透射电镜可见感染后的胎鼠大脑皮质细胞构筑破坏严重，细胞器均有不同程度损伤，并在胞核内出现病毒核酸、胞质内出现疱疹样病毒颗粒。结论HCMV AD169株具有感染体外培养的胎鼠大脑皮质细胞的能力，证实了HCMV可以跨种属感染，为临床研究HCMV中枢神经系统感染提供了实践依据。
[Abstract]:Objective to study the proliferative infection of human cytomegalovirus (HCMV) in fetal rat cerebral cortex cells in vitro and to explore the molecular mechanism of HCMV transspecies infection. To provide experimental basis for clinical study of HCMV central nervous system infection. Methods the purified HCMV AD169 strain was detected for viral infection by plaque forming unitu test. 4 脳 10 ~ (5) PFU/ml virus was inoculated into the cerebral cortex cells and human embryonic lung fibroblasts of Balb/c fetal mice. The cell cultures and their supernatants were collected on the 1st day after infection and the supernatants were collected on the 7th day after inoculation, and the following indexes were detected by different methods. To observe the cytopathic effect of HCMV on fetal rat cerebral cortex cytopathic cytopathic cytopathic effect and its pathological characteristics by inverted microscope. To detect the replication and proliferation of UL54 DNA, UL54 DNA and mRNAs in fetal rat cerebral cortex cells by PCR and RT-PCR. Taking 尾 -actin as internal parameter, Study on the change characteristics and trend of mRNA content: (3) using plaque formation test to detect the number of infective viruses when the effect of HCMV infection on fetal cortical cytopathic effect reaches ~ ~ ~) observing the infection of HCMV by transmission electron microscope (TEM). Changes of viral particles and ultrastructure in fetal rat cerebral cortex cells. Results 1) 2 days after inoculating HCMV AD169 strain with 4 脳 10 ~ 5 PFU/ml to fetal rat cerebral cortex cells, the cytopathic effect of virus was found. A few neurons and glial cells were swollen, round, intracellular granules increased and refractive index increased. On the 5th and 7th day, the number of cytopathic lesions increased gradually, and the degree of cytopathic lesions became more serious, such as axonal breakage, cell dissolution and even exfoliation. However, the HEL inoculated with the virus appeared on the first day, and with the prolongation of time, the number of cytopathic lesions increased and the pathological changes became more serious, which showed cell swelling, roundness, enhanced refraction and shedding. Relatively speaking, HEL was more sensitive to HEL and the cytopathic degree was higher. Both HCMV IEMUL83 and UL54 DNA were detected by PCR on the 5th and 7th day after inoculation of HCMV with fetal rat cerebral cortex cells. HCMV IEUL83 and UL54 mRNAs were detected at each time point by RT-PCR with 尾 -actin as the internal reference. The product sizes were 872 BP and 784 BP, respectively. The results of Bioscience image anlysis system showed that the mRNAs were 872 BP and 784 BP, respectively. With the prolongation of the time of HCMV infection in fetal rat cerebral cortex cells, the expression of HCMV-IEMV-UL83 and UL54 mRNA increased significantly. It was confirmed that HCMV was proliferative infection in fetal rat cerebral cortex cells. The results of plaque formation test showed that: about the 7th day, the expression of HCMV-IEMV-UL83 and UL54 mRNA in fetal rat cerebral cortex cells was increased. Round plaque appeared in the HEL cell layer inoculated with virus suspension. With the increase of infection days, the plaque became larger and the number increased. According to the formula, the virus infection amount was 3.8 脳 10 ~ 5 PFU / ml. 4) transmission electron microscope showed that the damage of cerebral cortex cell architecture was serious and the organelles were damaged in different degrees. Virus nucleic acid appeared in the nucleus and herpesvirus particles appeared in the cytoplasm. Conclusion HCMV AD169 strain has the ability of infecting fetal rat cerebral cortex cells cultured in vitro, which proves that HCMV can transspecies infection and provides a practical basis for clinical study of HCMV central nervous system 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】：2007
【分类号】：R373

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