巨细胞病毒感染小鼠对腹腔巨噬细胞功能的影响

巨细胞病毒（ＣＭＶ）在人群中的感染较为普遍，且多为隐性感染。为了观察ＣＭＶ感染对宿主免疫功能的影响，选择了机体重要的防御细胞－巨噬细胞作为研究对象。利用小鼠腹腔渗出细胞（ＰＥＣ）总数、巨噬细胞含量、ＦＣ花环形成率和肿瘤坏死因子（ＴＮＦａ）释放量检测鼠...     

抗钙调素抗体对抗体产生细胞分泌抗体功能的影响

以钙调素拮抗剂三氟拉嗪（ＴＦＰ）和环抱素Ａ（ＣｓＡ）为对照，观察抗钙调素（抗ＣａＭ）抗体对绵羊红细胞（ＳＲＢＣ）免疫的小鼠脾细胞及分泌抗巨细胞病毒（抗ＣＭＶ）单克隆抗体的杂交瘤细胞抗体分泌功能的影响。发现ＴＦＰ（０．１～１００μｍｏ／Ｌ）、ｃｓＡ（０．１～１００μ／Ｌ）明显抑制小鼠脾细胞与杂交瘤细胞分泌鼠Ｉｇ的功能，且此抑制作用随ＴＦＰ、ＣｓＡ剂量的增大而增强；与此不同，人和兔抗ＣａＭ对上述细胞分泌Ｉｇ有明显促进作用。     

住院患儿唾液排巨细胞病毒的调查

住院患儿唾液排巨细胞病毒的调查单玉君（上海市儿童医院，上海２０００４１）巨细胞病毒（ＣＭＶ）隐性感染在人群中普遍存在，初次感染大都发生在婴幼儿期。本实验室曾调查１～６月龄正常婴儿唾液ＣＭＶ阳性率达５７％，而我国先天性ＣＭＶ感染率仅０．５％～３．９９％...     

健康成人巨细胞病毒性肝炎

健康成人巨细胞病毒性肝炎北京佑安医院陈义森，林秀玉综述人巨细胞病毒（ＣＭＶ）感染极为普遍，ＣＭＶ可广泛分布于全身各脏器中引起细胞炎症反立，因年龄和免疫功能状态不同，临床病理表现和预后各异。肝脏ＣＭＶ感染多发生于儿童、孕妇及接受大量输血、脏器移植、体外...     

单细胞显微切割术在霍奇金淋巴瘤中的应用

目的：应用单细胞显微切割技术分离和研究霍奇金淋巴瘤（Ｈｏｄｇｋｉｎ‘ｓ ｌｙｍｐｈｏｍａ,ＨＬ）瘤细胞,并检测其是否存在人巨细胞病毒（ｈｕｍａｎ ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ,ＨＣＭＶ）感染。方法：采用单细胞显微切割技术从ＨＬ组织切片中获得其瘤细胞Ｒｅｅｄ－Ｓｔｅｒｎｂｅｒｇ／Ｖａｒｉａｎｔ （ＲＳ／Ｖ）细胞,提取ＤＮＡ后,利用ＰＣＲ扩增ＨＣＭＶ立即早期基因片段。结果：单细胞显微切割可将ＲＳ／Ｖ     

沈阳市献血员抗巨细胞病毒抗体调查

沈阳市献血员抗巨细胞病毒抗体调查方德强，王义武，吉耀华（中国医科大学附属第二医院，沈阳１１０００３）输血导致人巨细胞病毒（ＨＣＭＶ）感染，国内外均有报道，因此检测献血员中巨细胞病毒特异性ＩｇＭ抗体（ＨＣＭＶ－ｓＩｇＭ）对预防输血传播及骨髓移植的失败，...     

珠海地区小儿巨细胞病毒肺炎68例临床分析

对１８６名肺炎患儿血清中抗巨细胞病毒免疫球蛋白ＩｇＭ（ＣＭＶ－ＩｇＭ）进行了检测，提出了阳性６８例，占３６．５６％，对其与年龄，居住环境，合并症以及肺炎病情轻重的内在关系进行了探讨，发现感染ＣＭＶ的６８例中，围生期和〉３岁者分别占６１．５４％（２４／４８例）和４０．９１％（９／４８例）提示加强育龄妇女的保健，积极防治围生期和群聚儿童ＣＭＶ感染是降低婴幼肺炎发病率和死亡率的重要措施。     

原位杂交法检测柯萨奇B3病毒感染鼠各脏器内病毒核酸的动态分布

用柯萨奇Ｂ３病毒（ＣｏｘｓａｃｋｉｅｖｖｉｒｕｓＢ３，ＣＶＢ３）ｃＤＮＡ探针原位杂交法检测感染后１２ｄ内小鼠各脏器内病毒核酸的分布。于不同时期分别在心肌细胞、骨骼肌细胞、胰腺细胞、胸腺细胞、脾脏红髓细胞、直肠上皮细胞和肾小管上皮细胞中检测到病毒核酸。为ＣＶＢ３对多脏器的侵犯提供了直接的依据。     

柯萨奇B3病毒诱导小鼠单核-巨噬细胞产生肿瘤坏死因子和白细胞介素6

观察柯萨奇Ｂ３（ＣｏｘＢ３）病毒对小鼠单核－巨噬细胞（Ｍｏ／Ｍφ）的感染，及诱导白细胞介素６（ＩＬ－６）和肿瘤坏死因子（ＴＮＦ）的产生。发现ＣｏｘＢ３能够感染小鼠腹腔巨噬细胞，并释放感染性病毒颗粒；一定感染量病毒的感染，对Ｍｏ／Ｍφ的存活率无明显影响。在体内和体外实验中，ＣｏｘＢ３的感染均可明显诱导Ｍｏ／Ｍφ释放单核因子，在受染的Ｍｏ／Ｍφ培养上清液中及ＣｏｘＢ３感染急性期小鼠血浆和心肌匀浆液中均可测到ＩＬ－６和ＴＮＦ浓度明显升高。     

肾移植患者血清中多项免疫参数的动态观察

本文分析２０例无严重排斥反应的肾移植患者与正常人相比血中纤连素（Ｆｎ）增高，而补体成Ｃ３、Ｃ４含量均低，但Ｃ反应性蛋白（ＣＲＰ）阴性，血中肿瘤坏死因子（ＴＮＦ）水平与正常人近似。肾移植术后，短期内见有ＣＲＰ迅速而明显上升，而ＴＮＦ早期呈明显降低，随后二者均逐渐恢复正常，Ｃ３、Ｃ４和Ｆｎ虽然均有波动，但前二者仍低于正常，后者仍高于正常水平，特别值得注意的是移植前仅有少数病人可检出巨细胞病毒（ＣＭＶ）ＩｇＭ／ＩｇＡ抗体，但移植后２～４周所有病人ＣＭＶＩｇＭ／ＩｇＡ抗体均呈阳性。     

Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection

In this study, antiviral effect of black seed oil (BSO) from Nigella sativa was investigated using murine cytomegalovirus (MCMV) as a model. The viral load and innate immunity mediated by NK cells and Mφ during early stage of the infection were analyzed. Intraperitoneal (i.p.) administration of BSO to BALB/c mice, a susceptible strain of MCMV infection, strikingly inhibited the virus titers in spleen and liver on day 3 of infection with 1x10(5) PFU MCMV. This effect coincided with an increase in serum level of IFN-gamma. Although BSO treatment decreased both number and cytolytic function of NK cells on day 3 of infection, it increased numbers of Mφ and CD4(+) T cells. On day 10 of infection, the virus titer was undetectable in spleen and liver of BSO-treated mice, while it was detectable in control mice. Although spleen of both control and BSO-treated mice showed similar CTL activities on day 10 after infection, serum level of IFN-gamma in BSO-treated mice was higher. Furthermore, BSO treatment upregulated suppressor function of Mφ in spleen. These results show that BSO exhibited a striking antiviral effect against MCMV infection which may be mediated by increasing of Mφ number and function, and IFN-gamma production.     

Cadaveric skin allograft-associated cytomegalovirus transmission in a mouse model of thermal injury.

As a routine procedure to provide temporary coverage for burn wounds, cadaveric skin allografts have been used in patients with massive thermal injuries. In this study, CMV infection associated with skin grafting was investigated. Graft-associated CMV transmission was shown in a mouse model of thermal injury. Skins from mice 100 days after a nonlethal dose of murine CMV (MCMV) infection contained MCMV DNA and mRNA, although the virus was not isolated from these murine skins. When these skins were grafted to burned mice, the marked growth of MCMV was demonstrated in salivary glands. No viral growth was shown in the salivary glands of unburned mice or CMV sero(+) mice after grafting with these skins. When severe combined immunodeficient beige (SCID-beige) mice were used as recipients for CMV sero(+) skins, all mice died within 30 days after the grafting. Only 1 PFU/mouse of MCMV was shown to be 1 LD(50) in SCID-beige mice, while a 50% mortality rate was shown in normal unburned mice infected with 5 x 10(5) PFU/mouse of MCMV. This indicates that a very small amount of CMV contained in skins is sufficient to induce CMV infection in immunocompromised hosts. On the other hand, human CMV (HCMV) DNA and mRNA were detected by PCR analysis in 55% (DNA) and 33% (mRNA) of cadaveric skins, although the isolation of HCMV from cadaveric skin homogenates was not achieved in tissue cultures. CMV sero(-) patients with severe burn injuries may have a high risk for CMV infection associated with allografts of cadaveric skins.     

Alteration of host defense mechanisms by murine cytomegalovirus infection.

An animal model of a sublethal infection, utilizing murine cytomegalovirus (MCMV), was developed to determine whether immunological factors could contribute to the establishment of a persistent viral infection. Adult female C3H mice inoculated intraperitoneally with 10(5) plaque-forming units of MCMV developed splenomegaly 5 to 12 days after infection. Virus replicated to peak titers (10(3) to 10(6) plaque-forming units per g of tissue) in liver, spleen, lung, kidney, and salivary gland tissue during the acute phase of the infection (3 to 12 days); it then decreased to undetectable levels in all tissues except salivary gland. Serum interferon was detected as early as 12 h after infection, peaked at 36 h (1,093 U/ml), and was undetectable by 4 days after infection. MCMV-infected animals were hyporeactive to interferon induction with New castle disease virus on days 5 to 9 of the infection. Splenic lymphocyte reactivity to phytohemagglutinin and lipopolysaccharide was normal early during the course of the infection, was suppressed during the acute phase of the infection, and had returned to normal by day 18. These data indicate that several parameters of host defense are transiently suppressed during the course of a MCMV infection. The capacity of cytomegaloviruses to alter host resistance may be one factor that contributes to the establishment of a persistent infection.     

腺病毒载体介导的LacZ基因在神经前体细胞的转染和表达

为了观察含报告基因 L ac Z的重组 5型腺病毒载体 ( Ad5CMVLac Z)在神经前体细胞转染和表达的量效关系并探讨用该载体构建基因修饰细胞的可能性 ,本研究用不同滴度 ( 1× 10 3～ 1× 10 1 0 PFU/ml)的 Ad5CMVLac Z转染体外培养的 SD胎鼠(胎龄 12 d)海马神经前体细胞 ,用β-半乳糖苷酶 (β-gal)免疫组化反应检测转染效率。结果显示 :当病毒滴度为 1× 10 7时 ,转染率约为 5 0 % ,当滴度增加到 1× 10 1 0时 ,转染率达 10 0 %。Ad5CMVL ac Z在神经前体细胞的转染率具有滴度依赖性的量效关系。表明高滴度的 Ad5CMVLac Z成功地转染了大部分神经前体细胞 ,Lac Z基因也得到充分表达。提示神经前体细胞是该载体转染的适宜靶细胞 ,并有可能通过该载体转导目的基因 ,构建出基因修饰细胞     

Evaluation of continuous cell lines in antiviral studies with murine cytomegalovirus

Summary Cell culture systems were developed for rapid antiviral drug screening, using murine cytomegalovirus (MCMV) as an alternative to the slower growing human CMV. Since previous assay methods with MCMV employed mouse embryo fibroblasts (MEF cells), which are labor intensive to prepare and die off after 3–4 passages from primary culture, identification of virus-susceptible continuous cell lines was desirable. Three cell lines were found useful for assaying MCMV: C127I, SC-1, and 3T3. The antiviral agents acyclovir, ganciclovir, 5-fluoroarabinofuranosylcytosine, and 2-fluoro-2-deoxy-5-iodoarabinofuranosylcytosine were evaluated in the 3 continuous cell lines and in MEF cells. The 50% virus- or cell-inhibitory concentration values determined for each compound did not vary much from cell to cell. MEF cells were 10-fold more sensitive than the other cell lines to quantify virus from mouse organs, however. Virus propagated in 3T3 and SC-1 cells were as virulent to mice as salivary gland virus, whereas virus from MEF and C127I cells was more attenuated. Overall, C127I cells were judged to be the best for large scale antiviral screening in vitro, but MEF was the cell type of choice for titration of viruses from mouse organs and tissues.     

巨细胞病毒感染C57BL/6小鼠诱导NK细胞免疫应答的模型研究

目的研究小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染C57BL/6小鼠诱导自然杀伤(natural killer,NK)细胞免疫应答的最佳剂量和最佳时间。方法分别根据剂量和时间效应进行分组,剂量效应:无特定病原体(specific pathogen free,SPF)级8周龄C57BL/6雌鼠15只,根据MCMV滴度分为四组,未感染的0个蚀斑形成单位(plaque forming unit,PFU)组(3只)、2.5×10⁴PFU组(4只)、5.0×10⁴PFU组(4只)和10.0×10⁴PFU组(4只),随后常规饲养7天;时间效应:将8周龄C57BL/6雌鼠14只随机分为四组,分别为0天组(4只)、3天组(4只)、7天组(4只)、14天组(2只),建模0天腹腔注射5.0×10⁴PFU的MCMV感染各组小鼠。在感染后对应天数处死各组小鼠,取小鼠脾脏制成单细胞悬液,并用流式细胞术分析各种组NK细胞比例、Ly49H及颗粒酶B(granzyme B,GZMB)、NK细胞溶酶体相关膜蛋白-1(CD107a)和γ-干扰素(interferon-γ,IFN-γ)的表达。结果 MCMV感染7天后,与0PFU组相比,2.5×10⁴PFU组、5.0×10⁴PFU组和10.0×10⁴PFU组脾脏NK细胞占淋巴细胞的比例明显下降{(1.90±0.32)%比[(0.91±0.14)%,(0.62±0.16)%,(0.85±0.26)%],F=21.271,P<0.05},CD27⁺CD11b⁺NK细胞比例显著下降{(22.40±4.55)%比[(13.20±1.29)%,(10.78±1.21)%,(11.38±1.76)%],F=17.272,P<0.05},CD11b⁺NK细胞比例显著增加{(59.87±5.33)%比[(71.08±1.82)%,(74.25±1.95)%,(70.90±2.49)%],F=14.641,P<0.05)},CD43⁺KLRG1⁺NK细胞占NK细胞的比例增加{(39.40±5.73)%比[(77.00±0.67)%,( 81.23±2.21)%,( 76.63±7.36)%],F=55.282,P<0.05)},Ly49H⁺NK细胞亚群数量显著增加{(42.07±1.21)%比[(63.50±1.30)%,(63.58±3.71)%,(61.68±4.84)%],F=32.273,P<0.05)},组间差异具有统计学意义。在NK细胞功能方面,MCMV感染7天后,与0 PFU组相比,2.5×10⁴PFU组、5.0×10⁴PFU组和10.0×10⁴PFU GZMB的表达显著增加{(287.00±30.79)%比[(384.25±63.91)%,(529.75±66.08)%,(466.50±83.38)%],F=8.730,P<0.05},IFN-γ分泌减少{(36.00±5.33)%比[(4.88±3.35)%,(3.03±1.56)%,(3.61±2.18)%],F=87.663,P<0.05}。时间效应的比较结果显示,MCMV感染3天后,NK细胞CD107a和颗粒酶B表达与0天相比显著升高[(22.98±4.58)%比(6.32±0.75)%,(5969.25±1159.86)%比(788.50±88.17)%,F值分别为41.072和67.448,P值均<0.05],差异具有统计学意义。结论本研究表明,MCMV感染C57BL/6小鼠模型可选择的最佳感染剂量为5×10⁴PFU,最佳建模时间为感染后3天。     

Experimental inoculation of male mice with murine cytomegalovirus and effect on offspring

BACKGROUND: Murine cytomegalovirus (MCMV) was used to examine aspects of viral infection in male mice, and its possible transmission to their offspring. METHODS AND RESULTS: FVB/N mice inoculated intratesticularly with 5x10(5) plaque forming units (PFU) of MCMV, developed peritoneal haemorrhagic exudates, spleen hypertrophy and acute local infection. Infectiousness was detected until 15 days post-inoculation (D15 PI) in the genital organs, and virus DNA up to D35 PI. Testicular endothelial and Leydig cells were infected, and peritubular cells severely damaged. Spermatogenesis was affected, but neither germ cells nor Sertoli cells were infected. No virus was found in the epididymal epithelial cells. Viral DNA was detected in cells extracted from vas deferens samples until D15 PI. Neither infectious virus nor viral DNA were found in spermatozoa recovered from uterine fluid, fertilized oocytes, blastocysts, fetal tissues or newborn animals following the mating of infected males with uninfected females. CONCLUSIONS: MCMV harboured in the male genital organs was not transmitted to their offspring, even when mating occurred during the acute phase of CMV disease. Although the infection may have had an impact on spermatogenesis, fertility was not affected. These results do not support the hypothesis of conceptus MCMV infection by the fertilizing spermatozoon in natural conception.     

A focused salivary gland infection with attenuated MCMV: an animal model with prevention of pathology associated with systemic MCMV infection

While the salivary gland has been recognized as an important effector site of the common mucosal immune system, a useful model for studying anti-viral salivary gland immune responses in vivo and for exploring the role of the salivary gland within the common mucosal system has been lacking. Murine cytomegalovirus (MCMV) is a beta-herpesvirus that displays a strong tropism for the salivary gland and produces significant morbidity in susceptible mice when introduced by intraperitoneal (i.p.) inoculation. This study tested the hypothesis that MCMV morbidity and pathology could be reduced by injecting the virus directly the submandibular salivary gland (intraglandular (i.g.)), using either in vivo derived MCMV or the less virulent, tissue-culture-derived MCMV (tcMCMV). Peak salivary gland viral titers were completely unaffected by infection route (i.p vs. i.g.) after inoculation with either MCMV or tcMCMV. However, i.g. tcMCMV inoculation reduced viremia in all systemic tissues tested compared to i.p. inoculation. Furthermore, systemic organ pathology observed in the liver and spleen after i.p. inoculation with either MCMV or tcMCMV was completely eliminated by i.g. inoculation with tcMCMV. Cellular infiltrates in the salivary glands, after i.p. or i.g. inoculation were composed of both B and T cells, indicating the potential for a local immune response to occur in the salivary gland. These results demonstrate that a focused MCMV infection of the salivary gland without systemic organ pathology is possible using i.g. delivery of tcMCMV.     

重组腺病毒AdEGFP/hVEGF165快速构建及在骨髓移植小鼠体内的分布和表达效率

目的　研究增强型绿色荧光蛋白 (EGFP)标记的人血管内皮细胞生长因子 16 5(hVEGF16 5 )重组腺病毒载体的快速构建及在骨髓移植后小鼠体内的分布和表达效率。方法　利用细菌内质粒间同源重组法快速构建EGFP标记的重组腺病毒Ad EGFP hVEGF16 5 ;通过体外试验观察病毒的形态、滴度和安全性 ;经尾静脉注射 3× 10 8PFU的重组腺病毒给同基因骨髓移植BALB c小鼠 ,在不同时间检测重组腺病毒在小鼠体内分布和hVEGF16 5的表达。结果　通过细菌内质粒间同源重组法在短期内成功构建了复制缺陷型重组腺病毒载体Ad EGFP hVEGF16 5 ;纯化的病毒颗粒在电镜下成分均一 ,滴度可达 10 1 0 ～ 10 1 1 PFU ml。Hela细胞感染病毒后经多次传代未见细胞病变。借助于荧光显微镜在不同时期观测到小鼠心、肺、肝、脾、肾、小肠组织EGFP ;RT PCR分析和免疫组化染色显示脏器内有显著VEGF表达。各脏器未见明显毒性反应。ELISA法检测小鼠血浆中VEGF水平升高可达(86 6 6 7± 97 13)pg ml。结论　本研究结果证实细菌内同源重组法构建腺病毒载体具有高效、省时、省力的特点 ,并成功介导了hVEGF16 5基因在骨髓移植小鼠体内的安全、稳定表达 ,为今后在骨髓移植过程中开展基因治疗奠定了基础。     

Detection of murine cytomegalovirus (MCMV) DNA in skin using the polymerase chain reaction (PCR)

We used the polymerase chain reaction (PCR) and primers for the immediate early gene of murine cytomegalovirus (MCMV) to detect MCMV DNA in skin harvested from mice during acute infection. MCMV DNA was also detected in DNA extracted from spleen and salivary gland of MCMV-infected mice, but not in the skin, salivary gland, or spleen of uninfected, seronegative mice. Detection of MCMV DNA in skin provides direct evidence that skin can serve as a vehicle for transmission of MCMV. This observation is relevant to humans, such as burn patients, who receive skin allografts that may be infected with cytomegalovirus.