鼠巨细胞病毒（MCMV）对小鼠骨髓单个核细胞的潜伏感染研究

发布时间：2018-06-04 22:27

本文选题：MCMV + HCMV　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：人巨细胞病毒(Human Cytomegalovirus,HCMV)属于疱疹病毒科的β疱疹病毒亚科,是一种机会致病病原体,在人群中感染率达50%～80%。一般情况下,HCMV感染健康个体后无临床症状或者临床症状不明显,但是病毒可以以潜伏状态存在于宿主的外周血以及骨髓中,且能够通过血液制品或者固体器官移植进行传播。当机体免疫力低下时,潜伏的HCMV经活化后可导致致命性疾病。在骨髓移植患者中,HCMV感染所致死亡率仅次于移植物抗宿主病。鉴于HCMV潜伏感染对免疫力低下人群的严重危害,研究者对HCMV潜伏感染进行了深入的研究。由于HCMV感染具有严格的宿主要求,因此在整体水平的研究上,研究者多选择MCMV(Murine Cytomegalovirus)感染小鼠作为研究对象。 MCMV感染小鼠是HCMV研究的重要动物模型,主要是由于MCMV与HCMV的基因组之间具有线性相关性,MCMV含有的约200个开放阅读框与HCMV具有45.2%的相似性,所编码的蛋白有78个具有同源性。MCMV和HCMV在免疫缺陷或者免疫抵抗力不成熟的宿主体内都可导致严重的感染,并产生相似的临床症状,特别是对造血系统的影响方面。研究发现,MCMV感染小鼠后,小鼠造血系统细胞发生的改变主要表现为:外周血白细胞减少、体内血小板数量下降、骨髓中单核-巨核细胞前体细胞数量减少以及粒细胞-巨噬细胞集落形成单位(Granulocyte-Macrophage colony forming unit ,CFU-GM)和爆式红系集落形成单位(Erythroid burst forming unit,BFU-E)数量减少等,这些发现都与HCMV感染人类造血细胞非常类似。因此,用MCMV感染小鼠模型来研究HCMV感染对造血细胞群的效应,分析HCMV在造血细胞中原发和潜伏感染、活化以及重复感染的发病机制,对进一步研究HCMV潜伏感染以及潜伏再活化的机制有重要的理论和实际意义。研究表明,HCMV可潜伏在骨髓中,但对于究竟潜伏在骨髓的哪些细胞中尚没有确切的定论。为此,我们选择MCMV感染小鼠模型,开展了MCMV对小鼠骨髓细胞感染的研究,特别是在小鼠骨髓单个核细胞及其不同亚群细胞中的潜伏感染研究,主要工作包括: 1、MCMV对骨髓单个核细胞的潜伏感染研究。体外结果显示:MCMV体外感染分离培养的骨髓单个核细胞后,在单个核细胞中可检测到MCMV DNA以及MCMV立即早期基因的转录和蛋白的表达,未检测到早期基因的转录和蛋白的表达,且MCMV感染可以抑制骨髓单个核细胞集落的形成,提示MCMV可在体外潜伏感染骨髓单个核细胞;体内结果显示:MCMV病毒液直接经腹腔接种小鼠进行体内试验,在感染后的不同时间可以在小鼠的外周血以及骨髓中检测到MCMV DNA的存在,随着感染时间的增加,小鼠骨髓单个核细胞的数量也呈先下降后上升的趋势,说明MCMV体内感染小鼠在短期内可抑制小鼠骨髓的造血功能。 2、MCMV对骨髓单个核不同亚群细胞的潜伏感染研究。将骨髓单个核细胞进一步分选为不同的亚群细胞(包括lin~+、lin~-、lin~-cd117~+、lin~-cd117~-组分细胞),再进行MCMV感染。结果显示:在lin~+细胞可以检测到MCMV DNA的存在,并且可以检测到立即早期IE基因的转录产物和蛋白的表达,不能检测到早期E基因的转录和蛋白的表达;在MCMV感染的lin~-、lin~-cd117~+和lin~-cd117~-细胞中,未检测到立即早期IE基因和早期E基因的转录产物,间接免疫荧光也并未检测到有MCMV蛋白的表达;MCMV感染的lin~-cd117~+造血干/祖细胞的集落形成数量明显少于正常lin~-cd117~+造血干/祖细胞的集落数量,即MCMV感染对小鼠骨髓造血干/祖细胞的集落形成能力可以产生一定的抑制作用,且在MCMV感染的lin~-细胞中,具有干/祖细胞性质的CD117抗原的表达在MCMV感染之后比正常培养的细胞发生下调;在lin~-细胞中加入细胞因子(如GM~-CSF、rhEPO)或者诱导剂(如佛波酯)后可以使得MCMV在被诱导的lin~-细胞中表达立即早期蛋白IEP和早期蛋白EP,即细胞因子以及诱导剂可能会在诱导细胞分化过程中促进MCMV对细胞的感染。本研究结果证实,MCMV可以潜伏感染小鼠骨髓单个核细胞及其lin~+亚群细胞,并且在一定条件下可以在细胞中转录相应的基因和表达相关蛋白;含有造血干/祖细胞的lin~-细胞群对MCMV可能不易感,但是,lin~-细胞加入细胞因子或者诱导剂后可促进MCMV对细胞的感染,同时MCMV攻击可影响lin~-cd117~+造血干/祖细胞的功能和表型。我们的这些研究结果为进一步研究MCMV在骨髓中的潜伏与活化奠定了基础,具体的致病机制还有待进一步深入研究。
[Abstract]:Human cytomegalovirus (Human Cytomegalovirus, HCMV) belongs to the subfamily of herpes simplex virus in the herpetic family. It is an opportunistic pathogen. The infection rate is 50% to 80%. in the population. There is no clinical symptoms or clinical symptoms of HCMV infected healthy individuals, but the virus can be latent in the host's periphery. In blood and bone marrow, and can be transmitted through blood or solid organ transplantation. When the body's immunity is low, the latent HCMV can cause fatal disease. In bone marrow transplant patients, the mortality of HCMV infection is second only to graft-versus-host disease.
In view of the serious harm of HCMV latent infection to low immune population, researchers have studied the latent infection of HCMV in depth. Because of the strict host requirement of HCMV infection, the researchers chose the MCMV (Murine Cytomegalovirus) infected mice as the research object on the whole level of research.
MCMV infected mice are important animal models for HCMV research, mainly due to the linear correlation between the genome of MCMV and HCMV, about 200 open reading frames contained in MCMV have 45.2% similarities with HCMV, and the encoded proteins contain 78 homologous.MCMV and HCMV in the host body of immune deficiency or immune resistance. It can cause serious infection and produce similar clinical symptoms, especially on the effect on the hematopoietic system. After MCMV infection, the changes in the hematopoietic system of mice are mainly manifested in the decrease in peripheral blood leukocyte, the decrease in the number of platelets in the body and the decrease in the number of mononuclear cell precursor cells in the bone marrow and the decrease in the number of cells in the bone marrow and the decrease in the number of cells in the mononuclear cell precursor cells in the bone marrow. The number of granulocyte macrophage colony forming unit (Granulocyte-Macrophage colony forming unit, CFU-GM) and detonating erythroid colony forming unit (Erythroid burst forming unit, BFU-E) decreased, and so on. These findings are very similar to those of HCMV infected human hematopoietic cells. The effect of the cell group, the analysis of the pathogenesis of HCMV in the primary and latent infection of hematopoietic cells, activation and the mechanism of repeated infection, has important theoretical and practical significance to further study the mechanism of latent infection and latent reactivation of HCMV.
Studies have shown that HCMV can be latent in the bone marrow, but there is no definite conclusion about which cells which are latent in the bone marrow. For this reason, we choose the mouse model of MCMV infection and carry out the study of MCMV on the infection of bone marrow cells in mice, especially in the murine bone marrow mononuclear cells and the latent infection in different subgroups. The work includes:
1, MCMV's latent infection in bone marrow mononuclear cells. In vitro results showed that after MCMV infection was isolated and cultured bone marrow mononuclear cells, the transcription and protein expression of MCMV DNA and the immediate early gene of MCMV were detected in mononuclear cells, and the transcription and protein expression of the early gene were not detected, and the MCMV infection could be suppressed. The formation of the colony of bone marrow mononuclear cells indicates that MCMV can infect bone marrow mononuclear cells in vitro. In vivo, the results show that MCMV virus is inoculated into mice by intraperitoneal inoculation in vivo, and the presence of MCMV DNA in peripheral blood and bone marrow of mice can be detected at different time after infection, with the increase of time of infection, The number of bone marrow mononuclear cells in mice also decreased first and then increased. This indicates that MCMV infection in vivo can inhibit the hematopoietic function of bone marrow in mice in a short time.
2, MCMV studies the latent infection of different subgroup cells in bone marrow mononuclear cells. Bone marrow mononuclear cells are further divided into different subgroups (including lin~+, lin~-, lin~-cd117~+, lin~-cd117~- subcells) and then MCMV infection. The results show that MCMV DNA can be detected in lin~+ cells and immediate early I can be detected. The expression of the transcriptional products and proteins of the E gene could not detect the transcription of the early E gene and the expression of the protein; in the lin~-, lin~-cd117~+ and lin~-cd117~- cells infected with MCMV, the immediate early IE gene and the early E gene were not detected, and the indirect immunofluorescence was also not detected with the expression of MCMV protein; MCMV infection lin~-cd The colony formation of 117~+ hematopoietic stem / progenitor cells is significantly less than that of normal lin~-cd117~+ hematopoietic stem / progenitor cells, that is, MCMV infection can inhibit the colony formation ability of mouse bone marrow hematopoietic stem / progenitor cells, and the expression of CD117 antigen with stem / progenitor cell properties in the lin~- cells infected by MCMV is in MC MV infection is lower than normal cultured cells; adding cytokines (such as GM~-CSF, rhEPO) or inducers (such as phorbol ester) in lin~- cells can make MCMV express immediate early protein IEP and early protein EP in the induced lin~- cells, that is, cytokines and inducers may induce cell differentiation in the process of inducing cell differentiation. Into the MCMV infection of the cells.
The results of this study confirm that MCMV can infect murine bone marrow mononuclear cells and their lin~+ subgroup cells, and can transcribe the corresponding genes and expression related proteins in the cells under certain conditions, and the lin~- cell group containing hematopoietic stem / progenitor cells may not be susceptible to MCMV, but lin~- cells are added to the cytokines or inducers. It can promote the infection of MCMV to cells, and MCMV attacks can affect the function and phenotype of lin~-cd117~+ hematopoietic stem / progenitor cells. Our results have laid the foundation for further study of the latency and activation of MCMV in the bone marrow, and the specific pathogenesis remains to be further studied.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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