本文選題：MCMV + HCMV��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：人巨細(xì)胞病毒(Human Cytomegalovirus,HCMV)屬于皰疹病毒科的β皰疹病毒亞科,是一種機(jī)會(huì)致病病原體,在人群中感染率達(dá)50%～80%。一般情況下,HCMV感染健康個(gè)體后無臨床癥狀或者臨床癥狀不明顯,但是病毒可以以潛伏狀態(tài)存在于宿主的外周血以及骨髓中,且能夠通過血液制品或者固體器官移植進(jìn)行傳播。當(dāng)機(jī)體免疫力低下時(shí),潛伏的HCMV經(jīng)活化后可導(dǎo)致致命性疾病。在骨髓移植患者中,HCMV感染所致死亡率僅次于移植物抗宿主病。 鑒于HCMV潛伏感染對(duì)免疫力低下人群的嚴(yán)重危害,研究者對(duì)HCMV潛伏感染進(jìn)行了深入的研究。由于HCMV感染具有嚴(yán)格的宿主要求,因此在整體水平的研究上,研究者多選擇MCMV(Murine Cytomegalovirus)感染小鼠作為研究對(duì)象。 MCMV感染小鼠是HCMV研究的重要?jiǎng)游锬Ｐ?主要是由于MCMV與HCMV的基因組之間具有線性相關(guān)性,MCMV含有的約200個(gè)開放閱讀框與HCMV具有45.2%的相似性,所編碼的蛋白有78個(gè)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體內(nèi)都可導(dǎo)致嚴(yán)重的感染,并產(chǎn)生相似的臨床癥狀,特別是對(duì)造血系統(tǒng)的影響方面。研究發(fā)現(xiàn),MCMV感染小鼠后,小鼠造血系統(tǒng)細(xì)胞發(fā)生的改變主要表現(xiàn)為:外周血白細(xì)胞減少、體內(nèi)血小板數(shù)量下降、骨髓中單核-巨核細(xì)胞前體細(xì)胞數(shù)量減少以及粒細(xì)胞-巨噬細(xì)胞集落形成單位(Granulocyte-Macrophage colony forming unit ,CFU-GM)和爆式紅系集落形成單位(Erythroid burst forming unit,BFU-E)數(shù)量減少等,這些發(fā)現(xiàn)都與HCMV感染人類造血細(xì)胞非常類似。因此,用MCMV感染小鼠模型來研究HCMV感染對(duì)造血細(xì)胞群的效應(yīng),分析HCMV在造血細(xì)胞中原發(fā)和潛伏感染、活化以及重復(fù)感染的發(fā)病機(jī)制,對(duì)進(jìn)一步研究HCMV潛伏感染以及潛伏再活化的機(jī)制有重要的理論和實(shí)際意義。 研究表明,HCMV可潛伏在骨髓中,但對(duì)于究竟?jié)摲诠撬璧哪男┘?xì)胞中尚沒有確切的定論。為此,我們選擇MCMV感染小鼠模型,開展了MCMV對(duì)小鼠骨髓細(xì)胞感染的研究,特別是在小鼠骨髓單個(gè)核細(xì)胞及其不同亞群細(xì)胞中的潛伏感染研究,主要工作包括: 1、MCMV對(duì)骨髓單個(gè)核細(xì)胞的潛伏感染研究。體外結(jié)果顯示:MCMV體外感染分離培養(yǎng)的骨髓單個(gè)核細(xì)胞后,在單個(gè)核細(xì)胞中可檢測(cè)到MCMV DNA以及MCMV立即早期基因的轉(zhuǎn)錄和蛋白的表達(dá),未檢測(cè)到早期基因的轉(zhuǎn)錄和蛋白的表達(dá),且MCMV感染可以抑制骨髓單個(gè)核細(xì)胞集落的形成,提示MCMV可在體外潛伏感染骨髓單個(gè)核細(xì)胞;體內(nèi)結(jié)果顯示:MCMV病毒液直接經(jīng)腹腔接種小鼠進(jìn)行體內(nèi)試驗(yàn),在感染后的不同時(shí)間可以在小鼠的外周血以及骨髓中檢測(cè)到MCMV DNA的存在,隨著感染時(shí)間的增加,小鼠骨髓單個(gè)核細(xì)胞的數(shù)量也呈先下降后上升的趨勢(shì),說明MCMV體內(nèi)感染小鼠在短期內(nèi)可抑制小鼠骨髓的造血功能。 2、MCMV對(duì)骨髓單個(gè)核不同亞群細(xì)胞的潛伏感染研究。將骨髓單個(gè)核細(xì)胞進(jìn)一步分選為不同的亞群細(xì)胞(包括lin~+、lin~-、lin~-cd117~+、lin~-cd117~-組分細(xì)胞),再進(jìn)行MCMV感染。結(jié)果顯示:在lin~+細(xì)胞可以檢測(cè)到MCMV DNA的存在,并且可以檢測(cè)到立即早期IE基因的轉(zhuǎn)錄產(chǎn)物和蛋白的表達(dá),不能檢測(cè)到早期E基因的轉(zhuǎn)錄和蛋白的表達(dá);在MCMV感染的lin~-、lin~-cd117~+和lin~-cd117~-細(xì)胞中,未檢測(cè)到立即早期IE基因和早期E基因的轉(zhuǎn)錄產(chǎn)物,間接免疫熒光也并未檢測(cè)到有MCMV蛋白的表達(dá);MCMV感染的lin~-cd117~+造血干/祖細(xì)胞的集落形成數(shù)量明顯少于正常lin~-cd117~+造血干/祖細(xì)胞的集落數(shù)量,即MCMV感染對(duì)小鼠骨髓造血干/祖細(xì)胞的集落形成能力可以產(chǎn)生一定的抑制作用,且在MCMV感染的lin~-細(xì)胞中,具有干/祖細(xì)胞性質(zhì)的CD117抗原的表達(dá)在MCMV感染之后比正常培養(yǎng)的細(xì)胞發(fā)生下調(diào);在lin~-細(xì)胞中加入細(xì)胞因子(如GM~-CSF、rhEPO)或者誘導(dǎo)劑(如佛波酯)后可以使得MCMV在被誘導(dǎo)的lin~-細(xì)胞中表達(dá)立即早期蛋白IEP和早期蛋白EP,即細(xì)胞因子以及誘導(dǎo)劑可能會(huì)在誘導(dǎo)細(xì)胞分化過程中促進(jìn)MCMV對(duì)細(xì)胞的感染。 本研究結(jié)果證實(shí),MCMV可以潛伏感染小鼠骨髓單個(gè)核細(xì)胞及其lin~+亞群細(xì)胞,并且在一定條件下可以在細(xì)胞中轉(zhuǎn)錄相應(yīng)的基因和表達(dá)相關(guān)蛋白;含有造血干/祖細(xì)胞的lin~-細(xì)胞群對(duì)MCMV可能不易感,但是,lin~-細(xì)胞加入細(xì)胞因子或者誘導(dǎo)劑后可促進(jìn)MCMV對(duì)細(xì)胞的感染,同時(shí)MCMV攻擊可影響lin~-cd117~+造血干/祖細(xì)胞的功能和表型。我們的這些研究結(jié)果為進(jìn)一步研究MCMV在骨髓中的潛伏與活化奠定了基礎(chǔ),具體的致病機(jī)制還有待進(jìn)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊美芳,范駿;潛伏人巨細(xì)胞病毒的激活機(jī)制[J];國外醫(yī)學(xué)(流行病學(xué)傳染病學(xué)分冊(cè));2003年02期

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本文編號(hào)：1979093