【作者】 刘贵育；

【导师】 王明丽； 史百芬；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2004， 硕士

【摘要】 目的：本研究在用不同剂量人巨细胞病毒(human cytomegalovirus，HCMV)感染小鼠建立潜伏感染模型的基础上，观察HCMV在小鼠体内的主要潜伏部位和各潜伏部位HCMV基因组中长独特序列(UL)区第83个基因(UL83)载量以及再激活时相应部位UL83基因载量的变化，研究原发感染病毒量与潜伏病毒UL83基因载量的相关性，探讨再激活发生机率与潜伏感染UL83基因载量之间的关系，从而为临床预防免疫功能低下患者发生HCMV疾病寻找可靠诊断指标以及治疗和监测疗效提供新思路。方法：首先确定建立潜伏感染小鼠的HCMV剂量，再建立HCMV潜伏感染小鼠的模型。将60只6～8周龄SPF级BALB／c小鼠分为5组，每组12只，雌雄各半。A、B、C组为不同剂量病毒感染组：A组腹腔注射3.5×10~5PFU／ml HCMV 1.0ml／只；B 组腹腔注射3.5×10~5PFU／ml HCMV 0.75 ml／只；C组腹腔注射3.5×10~5 PFU／mlHCMV 0.5 ml／只。D组、E组为对照组：D组为DMEM对照组，腹腔注射DMEM液1.0 ml／只；E组为HF正常细胞对照组，腹腔注射正常HF细胞悬液1.0 ml／只。十四个月后将各组半数的小鼠处死，分别取小鼠肺、脑及外周血白细胞进行如下试验：(1)制备小鼠的肺、脑皮质匀浆及外周血白细胞进行病毒分离；(2)透射电镜下观察小鼠肺和脑组织有无病毒颗粒；(3)PCR检测HCMVUL83基因；(4)原位杂交检测HCMVUL83基因；(5)免疫荧光检测pp65蛋白表达；(6)实时荧光定量PCR检测HCMV UL83基因载量。同时对留下的小鼠进行免疫抑制干预，诱导潜伏HCMV的再激活。将每只小鼠称重，按150mg／kg的剂量给各组小鼠腹腔注射环磷酰胺，每隔6天注射一次，如此共注射3次，在最后一次注射后的第六天宰杀所有小鼠，取材及检测同前。结果：安徽医科大学硕士学位论文HCMV可以在小鼠体内建立潜伏感染状态并可被诱导再激活，而且，UL83基因是HCMV潜伏和再激活的标志性基因;肺、脑及外周血白细胞是HCMV的潜伏部位;各部位潜伏uL83基因载量有差异，肺uL83基因载量为8513士23leopies/10卜cells，与脑(250士1 seopies/lo6cells)和外周血白细胞(739士28c叩ies/IO6eells)有显著性差异(P<0.01)，故肺是主要的潜伏部位。A组原发感染病毒量最高，则各部位潜伏病毒DNA的量最高，从而发生再激活的机率最大;B组感染病毒量次之，则潜伏病毒DNA的量及发生再激活的机率也小于A组;C组感染病毒量最低，则潜伏病毒DNA的量及发生再激活的机率也是最低。潜伏时再激活UL83基因载量与发生再激活时UL83基因载量及发生再激活机率的相关性分析表明:二者呈正相关，相关系数及P值分别为10.02(P<0.05)和8.65(P<0.05)。结论:原发感染病毒量决定潜伏UL83基因载量，而潜伏UL83基因载量又决定再激活发生的部位和机率。高水平HCMV原发感染可导致高水平UL83基因潜伏，高水平UL83基因潜伏则可导致再激活发生的高风险。潜伏UL83基因载量与发生再激活的机率呈正相关。更多还原

【Abstract】 Objective: On the basis of establishment of a model for human cytomegalovirus (HCMV )latency in mice which were primary infected by different doses HCMV ,the study aimed at observing the latent sites and variation of the load of UL83 gene in corresponding sites in latency and reactivation, investigating the relationship between the burden of primary infection and the load of UL83 gene in latent infection,discussing the relationship between the risk of reactivation and the load of UL83 gene in latent infection. Based on the studies above ,to provide a new idea for diagnosis and therapy and prevention of HCMV disease in immunocompromised patients. Methods: Firstly, to determine the dose of HCMV resulting in latent infection in mice, and then establish a model for HCMV latency in mice. Sixty 6-8 week SPF BALB/c mice were divided into 5 groups, six female and six male mice in each group. The mice in group A were subjected to the intraperitoneal injection of 3.5 X 105 PFU/ml HCMV 1.0 ml per mouse. The mice in group B and group C were respectively infected in 3.5X 105 PFU/ml HCMV 0.75ml and 0.5 ml per mouse by intraperitoneal injection. Group D and Group E were normal control groups and the mice were respectively subjected to the intraperitoneal injection of DEME and HF 1.0 ml per mouse . After fourteen monthes, half of mice in per group were killed. The lungs and the brains and the blood leukocytes of mice were collected to be assayed as follows: (1) Human fetal fibroblasts(HF) grown as monolayers in cell culture were inoculated with lung or brain homogenate or blood leukocytes for virus isolation.(2)To look for HCMV like virus particle by electron microscopes.(3) The UL83 gene was detected qualitatively bypolymerase chain reaction(PCR).(4) The exsistence of UL83 gene was detected by in suit nucleic acid hybridization assay.(5) The expression of pp65 was assayed by immunofluorescent test.(6)The load of UL83 gene was measured by fluorescence quantitative real-time PCR. At the same time, the cyclophosphamide - a kind of immunosuppressant were injected into the intraperitoneum of the remained mice (150mg/kg) every six days for three times. Six days after the last injection, all mice were killed . The lungs and the brains and the blood leukocytes were collected to be assayed as test above . Results: HCMV can establish latency in mice ,which can be induced by using of immunosuppressant , moreover, UL83 gene is a marker gene in latency and reactivation.The lung and the brain and the blood leukocyte are the latent sites for HCMV, but the lung is the major site for latent HCMV. The load of latent UL83 in each latent site is remarkably different. There was 8513 31 copies/ 106cells UL83 gene in lung ,which is the highest.There were 250 + 18 copies/ 106cells and 739+28 copies/106cells UL83 gene in brain and blood leukocyte respetively. The difference is obviously (P<0.01) . The load of latent UL83 gene and the risk of recurrence in group A is the highest, as is level of primary infection of mice . The load of latent UL83 gene and the risk of recurrence in group B is lower than those in group A. The load of latent UL83 gene and the risk of recurrence in group C is the lowest. Our research showed that prolonged high-level virus replication during primary infection in mice led to a high load of latent viral DNA and a high incidence of recurrence when individual was immunosuppress. Correlative coefficent and P value were 10.02 (P< 0.05) ,8.65 (P< 0.05 ) .Conclusion: The level of primary infection defined the load of latent UL83 gene and the latter determined the latent sites and the risk of recurrence. A high load of latent UL83 gene can lead to a high incidence of recurrence from latency. The load of latent UL83 gene in HCMV latent infection mice had positive correlation with the risk of recurrence.更多还原