人巨细胞病毒感染对宿主细胞周期进程影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞DNA合成及细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0／G1期的人胚肺成纤维细胞（HEL），分别于感染后0、3．6、12、24、48、72、96h终止培养。用流式细胞术测定HCMV感染后细胞周期进程及DNA含量。用免疫蛋白印迹法（Western Blot）检测CyclinE、Cyclin A、Cyclin D1蛋白的表达。结果HCMV感染细胞后24h-96h，S期细胞明显增多，G2／M期细胞减少，至感染后96h，未发现有G2／M期细胞。感染后24h细胞保持2N DNA含量，全部感染细胞DNA含量在48h内开始升高，感染后72h多数细胞的DNA含量大于2N DNA含量。在正常对照细胞DNA含量没有增加。HCMV＋PAA组没有检测到DNA含量增加。HCMV感染接触抑制细胞12h时CyclinE蛋白被诱导，感染后24h出现Cyclin E峰值；HCMV不能诱导Cyclin A蛋白表达；CyclinD1在感染后24h开始下降。结论HCMV感染同步化于G0／G1期的细胞后，诱导CyclinE蛋白明显升高，激活CyclinE／Cdk2激酶，使细胞周期越过G1／S限制点，将细胞周期阻止于晚G1期。病毒感染未能激活细胞DNA合成，病毒感染后总DNA的增加由病毒DNA复制造成。     

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。     

人巨细胞病毒感染对宿主细胞周期蛋白表达影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0/G1期的人胚肺成纤维细胞（HEL）,分别于感染后0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h终止培养。用免疫蛋白印迹法（Western Blot）检测CyclinE、CyclinA、CyclinD1蛋白的表达。结果HCMV感染接触抑制细胞12 h时CyclinE蛋白被诱导,感染后24 h出现CyclinE峰值;HCMV不能诱导CyclinA蛋白表达;CyclinD1没有被诱导,且在感染后24 h开始下降。结论HCMV感染同步化于G0/G1期的细胞后,诱导CyclinE蛋白明显升高,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,将细胞周期阻止于晚G1期。     

细胞周期蛋白依赖性激酶cdk2和cdk4在前列腺增生和癌变组织中的表达

目的：探讨细胞周期蛋白依赖性激酶ｃｄｋ２和ｃｄｋ４在前列腺增生（ＢＰＨ）和前列腺癌（ＰＣ）的发生发展过程中作用及其与ＰＣＮＡ之间关系。方法：应用免疫组化ＳＰ法检测１８例正常前列腺（ＮＰ）、６２例ＢＰＨ和３３例ＰＣ组织中ｃｄｋ２、ｃｄｋ４和ＰＣＮＡ的表达。结果：前列腺上皮和间质组织中均见ｃｄｋ２和ｃｄｋ４表达。ＮＰ中两者表达均分别显著低于ＢＰＨ和ＰＣ;ＢＰＨ的上皮细胞中两者表达均分别显著低于ＰＣ,但ＢＰＨ的间质细胞中两者表达与ＰＣ相比均无显著性差异。ＢＰＨ和ＰＣ中ｃｄｋ２及ｃｄｋ４表达与ＰＣＮＡ指数均呈正相关。结论：ｃｄｋ２和ｃｄｋ４异常表达参与ＢＰＨ和ＰＣ的发生发展过程,其可能是通过改变细胞周期及促进细胞异常增殖而起作用的。     

人巨细胞病毒感染诱导宿主细胞凋亡的实验研究

人巨细胞病毒 (ＨＣＭＶ)活动性感染可以通过胎盘垂直传播 ,导致流产、畸形、死胎、新生儿神经系统及其他脏器的损害 ,已成为影响胎儿及新生儿生长发育的常见病原。现已证实 ,许多病毒性疾病 (如病毒性肝炎和艾滋病等 )的发生与凋亡失常有关〔1〕 。但目前有关ＨＣＭＶ与细胞凋亡的关系报道尚少。我们采用流式细胞技术对ＨＣＭＶ感染细胞进行细胞周期和凋亡分析 ,以探讨ＨＣＭＶ对宿主细胞的影响及机理。1　材料和方法1 1　病毒和细胞　ＨＣＭＶＡＤ16 9毒株由湖北省病毒研究所提供 ,按常规方法传代 ,滴定 ,其滴度为 10 3 ＴＣＩＤ5 0 0 .1…     

人巨细胞病毒感染诱导宿主细胞凋亡的实验研究

人巨细胞病毒 (ＨＣＭＶ)活动性感染可以通过胎盘垂直传播 ,导致流产、畸形、死胎、新生儿神经系统及其他脏器的损害 ,已成为影响胎儿及新生儿生长发育的常见病原。现已证实 ,许多病毒性疾病的发生与凋亡失常有关〔1〕。我们采用流式细胞技术对ＨＣＭＶ感染细胞进行细胞周期和凋亡分析 ,以探讨ＨＣＭＶ对宿主细胞的影响及机理。1　材料和方法1 1　病毒和细胞　ＨＣＭＶＡＤ16 9毒株由湖北省病毒研究所提供 ,按常规方法传代 ,滴定 ,其滴度为 10 3ＴＣＩＤ5 0 0 .1ｍｌ;人胚肺成纤维细胞 (ＨＥＬ)由同济医科大学附属同济医院儿科病毒室提供 ,…     

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。     

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。     

人巨细胞病毒感染对宿主细胞凋亡影响的研究进展

人巨细胞病毒（HCMV）属B疱疹病毒亚科，在人群中普遍感染。研究表明HCMV感染导致临床病症与细胞凋亡有密切关系，HCMV感染与细胞凋亡关系具有多样性和复杂性，在不同的阶段表现为不同的作用。研究HCMV与细胞凋亡的关系不仅有助于阐明HCMV感染细胞及细胞对其反应的分子机制和研发新的抗病毒制剂，而且能为HCMV引起临床疾病的治疗提供新的方案。     

人巨细胞病毒感染对宿主细胞凋亡影响的研究进展

人巨细胞病毒(HCMV)属β疱疹病毒亚科,在人群中普遍感染。研究表明HCMV感染导致临床病症与细胞凋亡有密切关系,HCMV感染与细胞凋亡关系具有多样性和复杂性,在不同的阶段表现为不同的作用。研究HCMV与细胞凋亡的关系不仅有助于阐明HCMV感染细胞及细胞对其反应的分子机制和研发新的抗病毒制剂,而且能为HCMV引起临床疾病的治疗提供新的方案。     

人巨细胞病毒感染对宿主细胞CyclinE/Cdk2激酶活性影响的实验研究

目的研究HCMV感染对Cdk2蛋白水平及对CyclinE/Cdk2激酶活性的影响。方法通过接触抑制使细胞同步化于G0/G1期,用MOI=5PFU/per cell HCMV AD169毒株感染人胚肺成纤维细胞（HEL）;用免疫沉淀,激酶活性分析法检测HCMV感染细胞Cdk2的活性。结果HCMV感染可引起CyclinE/Cdk2激酶的强烈激活,但HCMV感染并不诱导Cdk2蛋白丰度增加。结论HCMV感染G0/G1细胞,激活CyclinE/Cdk2激酶。使细胞周期越过G1/S限制点,进展至晚Gl期。     

Improving permissive infection of human cytomegalovirus in cell culture

Summary.  Changes in human cytomegalovirus (HCMV) titre occurring under different conditions were studied using plaque assay. No significant change in titre was found using primary embryonic fibroblasts or primary foreskin fibroblasts, or with the addition of dexamethasone to the medium. Significant increases in titre were found when standard cultures were pre-incubated in medium containing DEAE-dextran and/or calcium chloride. However, DEAE-dextran and/or calcium chloride had no significant effect on HCMV detection using the shell vial assay, possibly because enhancement affects permissive infection, but not surface expression of viral antigens. DEAE-dextran and calcium chloride can be included in the medium of standard cultures as a means of obtaining higher titres of HCMV, and are particularly useful for isolates that are difficult to culture. Received January 14, 2000/Accepted May 18, 2000     

Dynamics of T cell memory in human cytomegalovirus infection

Primary human cytomegalovirus (HCMV) infection of an immunocompetent individual leads to the generation of a robust CD4+ and CD8+ T cell response which subsequently controls viral replication. HCMV is never cleared from the host and enters into latency with periodic reactivation and viral replication, which is controlled by reactivation of the memory T cells. In this article, we discuss the magnitude, phenotype and clonality of the T cell response following primary HCMV infection, the selection of responding T cells into the long-term memory pool and maintenance of this memory T cell population in the face of a latent/persistent infection. The article also considers the effect that this long-term surveillance of HCMV has on the T cell memory phenotype, their differentiation, function and the associated concepts of T cell memory inflation and immunosenescence.     

Influence of congenital human cytomegalovirus infection and the NKG2C genotype on NK‐cell subset distribution in children

Human cytomegalovirus (HCMV) has been reported to reshape the NK‐cell receptor (NKR) distribution, promoting an expansion of CD94/NKG2C⁺ NK and T cells. The role of NK cells in congenital HCMV infection is ill‐defined. Here we studied the expression of NKR (i.e., NKG2C, NKG2A, LILRB1, CD161) and the frequency of the NKG2C gene deletion in children with past congenital infection, both symptomatic (n = 15) and asymptomatic (n = 11), including as controls children with postnatal infection (n = 11) and noninfected (n = 20). The expansion of NKG2C⁺ NK cells in HCMV‐infected individuals appeared particularly marked and was associated with an increased number of LILRB1⁺ NK cells in cases with symptomatic congenital infection. Increased numbers of NKG2C⁺, NKG2A⁺, and CD161⁺ T cells were also associated to HCMV infection. The NKG2C deletion frequency was comparable in children with congenital HCMV infection and controls. Remarkably, the homozygous NKG2C^+/+ genotype appeared associated with increased absolute numbers of NKG2C⁺ NK cells. Moreover, HCMV‐infected NKG2C^+/+ children displayed higher absolute numbers of NKG2A⁺ and total NK cells than NKG2C^+/? individuals. Our study provides novel insights on the impact of HCMV infection on the homeostasis of the NK‐cell compartment in children, revealing a modulatory influence of NKG2C copy number.     

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.     

巨细胞病毒感染后晚期mRNA表达与细胞病变相关性的研究

目的 研究巨细胞病毒(HCMV)感染后晚期mRNA的表达与致细胞病变作用(CPE)及感染细胞的超微结构变化。方法 用HCMV AD169株感染人胚肺成纤维细胞，通过半定量RT-PCR检测HCMV晚期mRNA的表达水平，同时动态观察CPE的改变，电镜观察细胞的超微结构变化。结果 HCMV晚期mRNA在感染后12h开始表达，随时间的延长水平逐渐升高；而CPE在48h后才出现，并逐渐加重。电镜下可见内质网早期囊腔扩张，晚期呈空泡变，线粒体肿胀，线粒体嵴数目减少，甚至缺失，感染晚期细胞核中有大量成熟待出壳的病毒核衣壳。结论 细胞超微结构变化与HCMV晚期mRNA表达密切相关，在病毒循环中起重要作用。晚期mRNA可作为观察治疗。HCMV活动性感染的临床疗效指标。     

Mechanisms of human cytomegalovirus infection with a focus on epidermal growth factor receptor interactions

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe diseases in immunocompromised individuals. It has a high prevalence worldwide that is linked with socioeconomic factors. Similar to other herpesviruses, HCMV has the ability to establish lifelong persistence and latent infection following primary exposure. HCMV infects a broad range of cell types. This broad tropism suggests that it may use multiple receptors for host cell entry. The identification of receptors used by HCMV is essential for understanding viral pathogenesis, because these receptors mediate the early events necessary for infection. Many cell surface components have been identified as virus receptors, such as epidermal growth factor receptor (EGFR), which is characterized by tyrosine kinase activity and plays a crucial role in the control of key cellular transduction pathways. EGFR is essential for HCMV binding, signaling, and host cell entry. This review focuses on HCMV infection via EGFR on different cell types and its implications for the cellular environment, viral persistence, and infection.