涎腺导管上皮细胞体外感染HCMV模型的建立

目的　建立涎腺导管上皮细胞体外人巨细胞病毒 (HCMV)感染模型。方法　用免疫细胞化学方法初步鉴定涎腺导管上皮细胞 (HSG)角蛋白 8(cytokeratin 8,CK8)和角蛋白 18(CK18)抗原的表达 ;观察HCMV感染HSG后导致的病变 ;观察宿主细胞中病毒颗粒的超微结构 ;用RT PCR及nest RT PCR检测HCMVIE1(即刻早期 1) /IE2基因的转录 ;用免疫细胞化学法检测HCMV感染细胞中IE1/IE2蛋白的表达。结果　呈上皮样贴壁生长的HSGCK8和CK18阳性表达 ;HCMV感染后 12小时 ( 12h .p .i.)即可见细胞病变 ,6 0h .p .i.CPE已极为明显 ,72h .p .i.绝大部分细胞出现病变。感染的HSG细胞在细胞核、内质网、细胞质中出现数量不等的 3种不同形态的疱疹病毒样颗粒。宿主细胞中可检测到HCMVIE1/IE2的转录以及IE1/IE2蛋白的表达。结论　成功建立了涎腺导管上皮细胞体外HCMV感染模型     

巨细胞病毒感染对动脉粥样硬化的影响

目的：研究巨细胞病毒(CMV)感染和动脉粥样硬化的关系，并探讨其可能机制。方法：通过一个大样本从血清流行病学(ELISA检测患者血清中抗CMV的IgG抗体)和分子生物学(PCR检测动脉粥样硬化病变中CMV特异性基因)方面探讨巨细胞病毒感染和动脉粥样硬化的关系，并检测CMV感染对内皮细胞趋化因子表达的影响。结果：动脉粥样硬化组血清中CMV的阳性率显著高于非动脉粥样硬化组(分别为82．2％和61．0％，P=0．02)；而且动脉粥样硬化斑块中HCMV基因出现率显著高于正常血管组织(13／15和2／7，P=0．01)；CMV感染还可上调内皮细胞：ECV-304表达MCP-1。结论：CMV感染参与动脉粥样硬化的形成和发生，这可能与CMV上调内皮细胞趋化因子表达有关。     

人巨细胞病毒基因时序表达对感染结局的影响

目的　探讨人巨细胞病毒 (HCMV)主要基因序列表达情况对其感染结局的影响。方法采用不同滴度HCMV感染传代培养的人胚肺成纤维细胞 ,建立HCMV梯度感染细胞模型。分别应用FQ PCR法测定感染细胞内IE基因拷贝数、免疫组化法联合计算机病理图文分析系统检测p5 2蛋白表达水平以及RT PCR法测定MCPmRNA转录水平 ,光镜观察致细胞病变作用 (CPE)、电镜检查细胞超微结构改变。结果　IE基因在病毒滴度最低的A组细胞内负荷量最低 (P <0 .0 5 ,P <0 .0 1) ;p5 2蛋白在病毒滴度较高的B、C组表达水平明显高于A组 (P <0 .0 1) ,其表达定位于受染细胞核内 ;仅在B、C组内检测到MCPmRNA。A组未检出细胞病变 ,B、C组细胞检测到随感染时间延长而加重的CPE及超微结构改变。结论　HCMV受染细胞内IE基因负荷量与病毒基因组表达启动密切相关 ,p5 2蛋白高效表达是病毒基因组完整表达的必要条件 ,MCPmRNA转录是活动性HCMV感染的标志。     

质粒介导的RNAi技术对截短HCMV IE2基因表达的抑制作用

目的 应用质粒介导的RNA干扰技术研究siRNA抑制截短HCMV IE2基因的表达的作用。方法 以pLL3．7质粒为模板，采用半套式PCR的方法扩增出含U6启动子的siRNA表达片段，连接pMD-T simple vector构建效应质粒表达重组体pMD-T-shRNARI和pMD-T-shRNAR2。同时采用RT-PCR方法从HCMV AD169株基因组中调取目的截短基因IE2，定向克隆到带有绿色荧光蛋白（GFP）作为报告基因的真核表达载体pEGFP-C1上，构建HCMVAD169株截短IE2基因重组表达质粒pEGFP-C1-IE2；用脂质体共转染效应质粒和靶基因表达重组体至Hep-2细胞中，荧光显微镜下观察效应质粒的RNA干涉作用，并观察RNA干扰的时效和量效改变。结果 三组质粒重组体构建成功，克隆到的2个shRNA表达载体对截短IE2基因的表达都有干涉作用。在与靶基因质粒量比为1：1和1：2时，其中pMD-T-shRNAR1 48～72h间干涉效应为100％，可完全封闭IE2的表达；pMD-T-shRNAR2使IE2处于极低水平表达，48～72h间干涉效应达到98．4％～99％。结论 通过质粒介导的RNAi技术能够有效地抑制HCMVIE2基因的表达，为治疗HCMV感染的药物开发提供了思路。     

HCMV IE2蛋白调控功能研究新进展

人巨细胞病毒（ human cytomegalovirus ， HCMV）属于β疱疹病毒亚科，是人类疱疹病毒中最大的一组病毒。在人群中，HCMV感染相当普遍，大多数感染呈临床隐性感染或潜伏感染；但在新生儿、免疫抑制或免疫功能低下的人群中则可引起临床显性感染，出现明显症状，甚至发生致死性疾病。而在孕妇，则可发生垂直传播，导致死胎、畸形以及婴幼儿神经系统发育障碍、智力低下等。 HCMV基因组全长超过240 kb的双链DNA，整个基因组含有250个开放阅读框（ ORF ）。在病毒感染过程中， HCMV基因的表达表现一定的时序性，可分为早早期（ IE ）、早期（ E）和晚期（ L）基因。病毒穿入细胞后， IE基因被宿主细胞因子激活并最早表达，编码两种重要的调控蛋白 IE1（ IE72）和 IE2（ IE86）。其中 IE2（ IE86）蛋白是从 HCMV 基因组的开放阅读框UL122上翻译过来的，是一种重要的反式激活因子并在HCMV感染中发挥了重要作用。已有研究表明，IE2（ IE86）能反式激活细胞周期蛋白cyclinE启动子，并能延缓宿主细胞进入S期［1-2］。近年有资料证实，IE2（ IE86）调节许多与控制细胞周期相关的因子。 IE2（ IE86）是一种重要的病毒蛋白质，而缺少IE2（ IE86）的HCMV子代突变体不能进行复制。下面主要就IE2（ IE86）蛋白对病毒蛋白表达调控的作用作一综述。     

大蒜新素对巨细胞病毒感染细胞周期蛋白基因表达谱的影响

目的 探讨人巨细胞病毒(human cytomegalovirus，HCMV)对感染细胞周期蛋白基因表达谱的影响及大蒜新素的干预效应。方法 利用基因芯片技术对比分析大剂量大蒜新素处理正常细胞和感染细胞(MOI=2．5)72h以及同期培养的感染和正常细胞周期相关蛋白基因(239条)表达谱的变化。结果 感染细胞有24条(10．04％)基因发生有意义的差异性表达；其中9条表达增加，15条表达降低。大蒜新素对ll条HCMV感染所致表达改变的基因无影响；使HCMV感染下调作用消除的基因有ll条；使HCMV感染上调作用消除的基因有2条。另外，药物对感染细胞和正常细胞基因表达均有影响的有2条；仅引起正常细胞基因表达改变的有3条。结论 HCMV感染使部分细胞周期相关蛋白基因差异性表达可能是HCMV导致宿主细胞周期紊乱的原因之一。大蒜新素至少可使HCMV所致13条基因差异性表达作用消除，提示药物干预了病毒对细胞周期的影响。     

UU和HCMV感染及细胞因子与输卵管妊娠的关系研究

本文研究目的是应用聚合酶链反应 (PCR)技术 ,检测 2 1份输卵管妊娠患者宫颈分泌物及输卵管液中解脲支原体 (UU)及巨细胞病毒 (HCMV) ,用ELISA法检测输卵管液中IL - 6、IL - 8,研究UU、HCMV感染和细胞因子变化与输卵管妊娠的关系。结果显示 :输卵管妊娠组宫颈分泌物和输卵管液中UU、HCMV检出率显著高于正常妊娠组 ,两者有显著性差异 (P <0 .0 5 ) ;输卵管妊娠组宫颈分泌物与输卵管液中UU、HCMV检出率比较无显著性差异 (P >0 .0 5 ) ;输卵管妊娠组输卵管液中IL- 6、IL - 8较正常妊娠组有极显著性差异 (P <0 .0 0 1)。结论 :输卵管妊娠与UU、HCMV感染和细胞因子变化有密切关系 ,与生殖道感染有密切关系。     

冠心病患者动脉粥样硬化与人巨细胞病毒检测结果的相关性分析

目的 观察人巨细胞病毒(HCMV)在冠心病患者动脉粥样硬化发病过程的相关性.方法 选取我院2014年8月至2016年4月心内科收治的动脉粥样硬化患者136例为观察对象,其中冠心病(CHD)患者103例,非CHD患者33例.使用多普勒超声对颈动脉内膜—中层厚度(IMT)进行监测,酶联免疫吸附试验法对血清HCMV-IgM进行检测.结果 HCMV感染组心肌梗死发生几率明显高于非HCMV感染组.HCMV感染组冠状动脉多支病变的发生几率亦显著高于非HCMV感染组.病变过程中HCMV感染和患者颈动脉IMT的相关性发现:HCMV感染患者颈动脉IMT(1.31 ±0.27) mm,非HCMV感染(1.14±0.22)mm,P＜0.05,差异有统计学意义;HCMV感染组的MMP-9以及软斑组织水平明显高于非HCMV感染组.将HCMV感染作因变量,将其他因素作自变量,筛选出3个影响因素,即:颈动脉IMT、MMP-9、软斑组织.结论 冠心病动脉粥样硬化与HCMV感染呈正相关,作为其影响因素可能会增加HCMV感染的几率.     

人巨细胞病毒感染对小鼠脑组织同源框基因表达的影响

目的　研究小鼠脑组织同源框基因 (homeoboxgene ,Hox)的表达状态及人巨细胞病毒(humancytomegalovirus ,HCMV)感染对小鼠脑组织Hox基因表达的影响 ,以探讨HCMV致畸的分子机制。方法　昆明系小鼠 4 8只 ,随机分为 :实验对照组 (16只 )和病毒感染组 (32只 ) ,病毒感染为脑内注射。在感染后 7、15、30、6 0d分别以病理学方法观察病理损伤 ,以免疫组化方法检查HCMV LA(latean tigen) ,PCR检测小鼠脑组织中HCMV DNA。在建立HCMV脑部感染的小鼠模型基础上 ,用RT PCR测定Hox基因在病毒感染组和实验对照组小鼠脑组织中的表达 ,并用同位素标记的Hox寡核苷酸探针进行Northernblot检测相应的表达状况。结果　 (1)接种HCMVAD16 9株的小鼠脑组织发生了病理改变 ,免疫组化和PCR方法在神经细胞内查到了HCMV LA和DNA ;(2 )RT PCR和Northernblot发现 :对照组的小鼠脑组织表达Hoxa2、Hoxb1和Hoxb2 ,但不表达Hoxb5、Hoxb8;HCMV感染后 ,小鼠脑组织被诱导表达Hoxa1,与对照组比较差异有显著性意义 (P <0 .0 1) ,且于感染后的 15d达到高峰 ,而Hoxb1的表达下调 (P <0 .0 5 ) ;Hoxa2和Hoxb2无明显变化 ;Hoxb5和Hoxb8仍旧不表达。结论　HCMVAD16 9株可感染小鼠神经系统。HCMV感染可诱导小鼠神经系统发育基因Hox基因表达改变 ,这对     

巨细胞病毒对细胞周期的影响及其机制

HCMV感染对细胞周期有很大的影响，处于不同细胞周期的细胞感染HCMV，皆可导致细胞周期的改变。在G0期及G1期感染HCMV可导致细胞周期进展到了G1/S限制点，在S期细胞感染HCMV可导致细胞周期受阻。细胞将不能进行有丝分裂，HCMV影响细胞周期主要是其极早期蛋白IE1-72及IE-86的作用，以IE2-86为主。其机制包括：使CyclinE的转录活性增加；把Cdk2从细胞浆转移到细胞核；细胞核中CKIs水平的下降。     

High prevalence of latently present cytomegalovirus in arterial walls of patients suffering from grade III atherosclerosis.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in arterial walls of patients with grade III and with maximally grade I atherosclerosis by dot blot and in situ DNA hybridization and by polymerase chain reaction (PCR) using probes and primers derived from immediate early (IE) and late (L) genomic regions. The presence of the complete viral genome could be demonstrated by both dot blot DNA hybridization and PCR. IE mRNA but not L mRNA could be demonstrated by in situ DNA hybridization, indicating the presence of latent CMV in the human arterial wall. By PCR 90% of the samples obtained from atherosclerotic patients were shown to contain viral nucleic acids as compared to 53% of patients with maximally grade I atherosclerosis, thus substantiating a role for this virus in the pathogenesis of atherosclerosis.     

Detection of human cytomegalovirus in the atherosclerotic cerebral arteries in Han population in China

The association of atherosclerosis (AS) and Human cytomegalovirus (HCMV) infection was studied. AS plays an important role in the brain stroke and HCMV infection is supposed to be involved in the process of atherosclerotic formation. The presence of HCMV DNA and antigens was examined in the internal carotid arteries collected from 35 patients with ischemic stroke and from 20 patients from the control population. All patients belonged to the ethnic Han population in China. Three methods, immunohistochemistry (IHC), hybridization in situ (HIS), and PCR were used to detect the HCMV immediate early (IE) and late (L) antigens as well as viral DNA in vessel walls. Levels of HCMV IE gene/protein were significantly higher in the stroke group than in control group detected by the three methods (IHC 34.3% vs. 10.0%; HIS 40.0% vs. 10.0; PCR 60.0% vs. 30.0%). However, there was no significant difference in the levels of HCMV L gene/protein between these two groups of patients (IHC 11.4% vs. 5.0%; HIS 11.4% vs. 10.0%; PCR 20.0% vs. 20.0%). We concluded that the presence of HCMV IE antigen and HCMV DNA in the vessel wall was associated with the pathological process of AS formation. Key words: Human cytomegalovirus; atherosclerosis; internal carotid arteries; ischemic stroke.     

The presence of cytomegalovirus nucleic acids in arterial walls of atherosclerotic and nonatherosclerotic patients.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in abdominal aortas and femoral arteries of patients with and without atherosclerosis by dot blot and in situ DNA hybridization using a DNA probe derived from immediate early genomic regions. Viral antigens could not be detected by immunohistochemistry and infectious virus could not be recovered from the arterial wall by virus isolation techniques. The high percentage (55%) of vascular wall specimens containing CMV nucleic acids, in atherosclerotic as well as in control material and the location of CMV-containing cells in arteries without gross changes indicative of atherosclerosis suggest that the human arterial wall may be a site of latency for this virus.     

The possible role of human cytomegalovirus (HCMV) in the origin of atherosclerosis.

BACKGROUND: The biological properties of some herpesviruses such as the ability of latent persistency in the host cells and the presence of viral DNA in atherosclerotic lesions, suggest the possible role of herpesviruses in the development of atherosclerosis. Although many authors proved the presence of viral DNA in arterial wall tissue, the role of herpesviruses in the origin and progress of atherogenesis still remains unclear. OBJECTIVES: The aim of this study was to confirm the presence of viral DNA in arterial wall and to associate the presence of these viruses with the development of atherosclerosis in patients with ischemic heart disease (IHD). STUDY DESIGN: A possible role of HCMV, EBV and HHV6 in the development of atherosclerosis was tested in 244 IHD patients and 87 coronarographically negative controls. The presence of viral DNA in aortic and venous walls, as well as in a peripheral blood samples was tested by the use of polymerase chain reaction (PCR) accompanied by, immunological tests for anti-virus antibodies IgM and IgG types for all experimental groups. RESULTS: The genomic DNA of HCMV was found in 76 and 59%, DNA of EBV in 59 and 50%, and DNA of HHV6 in 0.08 and 0.0%, of arterial walls of IHD patients and non-ischemic control group, respectively. No viral DNA was found in venous samples. Significant association (P < 0.01) has been proved between CMV infection and IHD. CONCLUSIONS: Our results suggest that HCMV and EBV can be found in the arterial wall, so that the arterial wall could be a potential site of persistency of those viruses. We also proved a significant association between the presence of HCMV DNA in aortic walls and atherosclerosis. Despite of the high genetic and biological similarity between CMV and HHV6 no substantial role of HHV6 in atherosclerosis has been proved.     

CMV and transplant-related coronary atherosclerosis: an immunohistochemical, in situ hybridization, and polymerase chain reaction in situ study.

Accelerated graft coronary atherosclerosis is the main obstacle to long-term survival in patients who have had a heart transplant. A possible involvement of the human cytomegalovirus (HCMV) in this type of coronary atherosclerosis has been postulated by many authors but has not been definitively demonstrated. In an attempt to clarify the role of HCMV infection in the pathogenesis of this complication, we looked for in situ antigens or DNA of HCMV in 30 coronary artery segments obtained at necropsy from patients who had undergone orthotopic cardiac transplantation at the S?o Paulo Heart Institute. We tried to correlate these HCMV markers with the presence of inflammation and/or atherosclerosis in histologic sections. The patients were grouped as follows: GI, less than 170 days of graft survival and absent/mild atherosclerosis; GII, more than 170 days of graft survival and absent/mild atherosclerosis; GIII, more than 170 days of graft survival and severe/moderate atherosclerosis (170 days was the shortest graft survival time associated with atherosclerosis). The search for HCMV genome and antigens in the coronary artery sections was performed using immunohistochemistry, in situ hybridization, and polymerase chain reaction in situ techniques. Immunohistochemistry and in situ hybridization revealed no evidence of HCMV in all 30 cases. Polymerase chain reaction in situ revealed scarce HCMV-positive lymphocytes in two cases (one each from GI and GIII) located in the adventitial layer. These findings preclude a direct role for the HCMV in the pathogenesis of accelerated graft coronary atherosclerosis. However, the possibility of an indirect effect of the virus, such as an immune-mediated inflammatory response by the host that increases the expression of histocompatibility antigens, leading to tissue injury, cannot be excluded.     

The immediate early genes of human cytomegalovirus upregulate expression of the cellular genes myc and fos.

Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (IE1 and IE2) can alter the regulation of the cellular immediate early genes (c-fos and c-myc). Plasmid constructs containing the promoter-regulatory regions c-myc or c-fos upstream of the reporter gene, chloramphemicol acetyl transferase, were co-transfected into T cells (Jurkat cells), monocytes/macrophages (THP-1 cells), or human fibroblast cells with plasmid constructs containing the promoter-regulatory region of the HCMV IE genes upstream of the bona fide IE1, IE2 or IE+2 genes; a plasmid that contained no IE coding region was used as a control. These studies show that both products of the HCMV IE genes markedly upregulated expression of the cellular c-fos and c-myc genes. The viral effects of individual proteins (IE1 or IE2) were dependent both on the promoter-regulatory region of the cellular gene and the cell type. In all cells, the combination of IE1 and IE2 further upregulated both cellular genes, suggesting a synergistic effect of IE1 with IE2. Both of the c-myc promoters (P1 and P2) were up-regulated by the HCMV IE gene products. IE1 and IE2 also upregulated the cells' endogenous c-myc and c-fos genes, as determined by amounts of the respective mRNAs. These studies show that HCMV can markedly alter cellular IE gene expression and that the effects of HCMV IE1 and IE2 proteins are dependent both on the promoter-regulatory region of the cellular gene and the type of cell in which the interaction occurs.     

Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX

The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes.     

人巨细胞病毒对小鼠宫颈的诱癌作用

用眼科镊将浸透了病毒母液的明胶海绵块塞入小鼠阴道，直抵宫颈的办法，将紫外线灭活的人巨细胞病毒（ＨＣＭＶ）投给昆明种杂交小鼠。约３１周后，有１５０％小鼠诱发了实验性宫颈癌，另一组加用巴豆油促癌剂，小鼠宫颈癌发率为１８８％，对照组癌发率为０。ＨＣＭＶ诱癌组１２例小鼠宫颈癌组织ＨＣＭＶＩＥ（ｉｍｍｅｄｉａｔｅｅａｒｌｙ）抗原均为阳性，血清ＨＣＭＶＩＥ抗体几何平均滴度为１∶１６７２，对照组以上两个指标均为阴性。ＨＣＭＶ诱癌组小鼠外周血淋巴细胞ＡＮＡＥ阳性率为３８９±６８％，对照组为６７８±８０％。