汉滩病毒PS-6株的分离及其生物学性状研究

目的 从病人血清分离汉坦病毒Ⅰ型毒株,为双价疫苗的配伍提供更好的候选株。方法 从陕西ＨＦＲＳ患者血清,按常规法分离汉坦病毒。用系列单克隆重抗体、空斑减少中和试验、ＲＴＰＣＲ及部分核苷酸序列分析等检定,确证所分离毒株的血清（或基因）型别。结果 从１７份ＨＦＲＳ患者血清中成功地分离到１株汉坦病毒,定名为ＰＳ－６株。经上述方法全面鉴定确定其为汉滩病毒（即Ⅰ型病毒）。该毒株有较强的繁殖力（毒力）及较好的抗     

汉滩病毒感染血管内皮细胞引起TLR4表达上调

目的 探讨汉滩病毒感染血管内皮细胞后,Toll样受体(TRY)分子的表达变化,为抗感染免疫和致病机制研究提供重要资料.方法 分别用LPS,CL097,Poly I:C以及汉滩病毒76-118刺激血管内皮细胞,6 h后提取总RNA,进行反转录获得cDNA,再分别用各自的引物进行PCR反应,产物用1%琼脂糖凝胶电泳分析,并用间接免疫荧光测定汉滩病毒感染血管内皮细胞后TLR4的表达.结果 汉滩病毒刺激感染后,血管内皮细胞TLR2、TLR4转录水平增高,TLR3转录水平降低,细胞膜表面的TLR4表达上调.结论汉滩病毒76-118感染血管内皮细胞后,可以引起TLRs分子转录水平发生改变,其中TLR4表达上调,固有免疫参与了汉滩病毒致病过程.     

汉滩病毒感染中的抗体依赖性增强作用的初步研究

在某些病毒的感染过程中,存在着抗体依赖性增强ＡＤＥ作用,即特异性抗体的存在可增强病毒的感染１,这是某些病毒疫苗免疫保护效果不好及某些病毒病的发病机制之一。汉滩病毒是人类肾综合征出血热ＨＦＲＳ的病原,机体对该病毒感染的免疫以体液免疫为主。目前评价ＨＦＲＳ疫苗接种效果的主要指标是特异性抗体的产生及其滴度高低,因此,采用特异性单克隆抗体ｍＡｂ治疗急性期ＨＦＲＳ患者也具有明显的疗效２。但另一方面,也有文献报道,在汉滩病毒感染实验中,曾观察到似有ＡＤＥ现象发生３。为进一步探讨在ＨＦＲＳ感染中是否存在ＡＤＥ…     

带有GFP标签的汉滩病毒糖蛋白表达载体的构建

目的:构建带有绿色荧光蛋白(GFP)标签的汉滩病毒(HTNV)包膜糖蛋白(GP)真核表达载体,并进行初步表达鉴定.方法:以peGFP-C1质粒为模板扩增GFP片段,将其插人真核表达载体pFLAG-CMV3中,构建成可表达带有GFP标签分泌型蛋白的载体pFLAG-CMV3-GFP.扩增HTNV-G1、HTNV-G2,分别插入上述载体,转染293T细胞,采用检测GFP标签蛋白的夹心ELISA法检测转染细胞培养上清中分泌型重组蛋白的含量.结果:酶切及测序证实带有GFP标签的HTNV-G1、HTNV-G2表达载体构建成功,分别命名为pFLAG-G1-GFP/pFLAG-G2-GFP,转染细胞的培养上清中可以检测出带有GFP标签的融合蛋白.结论:成功构建了带有GFP标签HTNV-GP的重组质粒并成功表达,为深入研究HTNV感染后机体针对HTNV-GP的特异性免疫应答规律奠定了实验基础.     

山茱萸总甙对HUVEC表达ICAM-1、CD44的影响

血管内皮细胞的活化在炎症疾病过程中起着重要作用 ,活化的血管内皮细胞可以表达粘附分子如ＩＣＡＭ 1和ＣＤ4 4 ,使中性粒细胞和淋巴细胞与其粘附、穿越内皮而进入炎症部位 ,研究表明中性粒细胞可通过其膜表面表达的配体与ＩＣＡＭ 1相互作用而粘附于血管内皮细胞的表面 ,ＣＤ     

RT-PCR检测不同人群庚型肝炎病毒感染

为了解成都地区ＨＧＶ感染情况 ,我们采用逆转录 聚合酶链反应 (ＲＴ ＰＣＲ)对职业献血员、健康自然人群和急慢性肝炎病人进行了检测。1　材料和方法1.1　对象　成都职业献血员 16 5名 ,各项体检均合格。成都健康自然人群 40 6名 ,Ａ～Ｅ肝炎血清标志均阴性 ,ＡＬＴ正常。肝炎病人包括急性甲型肝炎 32人 ,急性乙型肝炎 87人 ,慢性乙型肝炎 95人 ,慢性丙型肝炎 2 3人 ,非Ａ～Ｅ肝炎18人 ,这些病人均用酶联免疫吸附试验(ＥＬＳＡ) (上海科华试剂 )测定血清病毒抗原 /抗体证实。1.2　ＨＧＶ引物　据 5′ ＮＣＲ和ＮＳ5α区设计引物和捕获探针[…     

用RT-PCR技术检测hSIM基因的表达

ｈＳＩＭ基因（ｓｉｎｇｌｅ－ｍｉｎｄｅｄｇｅｎｅ）是新近发现的位于人类２１号染色体与胚胎细胞分化有关，并在人一些器官系统形成过程中起重要作用的基因。由于人基因组ｈＳＩＭ基因及ｈＳＩＭ基因ｃＤＮＡ均未克隆，研究ｈＳＩＭ基因表达对阐明该基因功能具有重要意...     

肾综合征出血热病人恢复期血清对汉滩病毒感染体 …

目的 用体外培养的昆明步鼠胚胎脑皮质神经元,感染汉滩病毒后,加入肾综合征出血热（ＨＦＲＳ）病人恢复期血清,观察病毒感染神经元后细胞的变化以及ＨＦＲＳ病人恢复期血清对神经元的保护作用。方法 将病毒感染的神经元加入不同浓度的ＨＦＲＳ病人恢复期血清（同时设立阴性血清对照组及正常细胞对照组和感染病毒对照组）,使用ＭＴＴ比色试验检测神经元存活情况。结果 各组神经元活性的ＭＴＴ比色试验显示,病毒感染组与未感染     

病毒性肝炎HGV-RNA的RT-PCR检测及血清学研究

庚 型肝炎病毒 (ＨＧＶ)是近年发现的新型肝炎病毒 ,能够引起非甲 非戊型肝炎 ;ＨＧＶ广泛存在于输血后肝炎及献血员中。本文应用ＲＴ ＰＣＲ技术对我市 1 996年 4月～5月间急性肝炎流行期间的 441例患者血清进行ＨＧＶ ＲＮＡ检测 ,并观察其肝损害情况 ,现报告如下。1　资料与方法1 .1　标本来源随机采集 1 996年 4月～ 5月间入院的急性肝炎患者血清 441例 ,病原学分型标准按 1 990年上海第六届全国病毒性肝炎会议修订的“病毒性肝炎防治方案”。1 .2　仪器和试剂ＨＧＶ ＲＮＡ测定采用ＲＴ ＰＣＲ法 ,试剂由北医大肝病研究所北京肝炎试剂…     

柯萨基B3病毒感染增强单个核细胞与心脏微血管内皮细胞的粘附

目的：研究柯萨基Ｂ３病毒（Ｃｏｘ Ｂ３）感染对内皮细胞表面ＩＣＡＭ－１表达的诱导作用,及对内皮细胞与单个核细胞粘附的影响,探讨病毒性心肌炎心肌细胞免疫损伤的机理。方法：采用流式细胞仪分析内皮细胞ＩＣＡＭ－１表达;粘附试验和单克隆抗体抑制试验观察单个核细胞与内皮细胞的粘附。结果：１００和４００ ＴＣＩＤ５０／ｍｌ Ｃｏｘ Ｂ３感染内皮细胞２４和４８ｈ,均可显著增加单个核细胞与内皮细胞的粘附（Ｐ〈０．０     

汉滩病毒核衣壳蛋白C-端T细胞表位鉴定

目的　鉴定汉滩病毒核衣壳蛋白 (HTNVNP)C 端T细胞表位 ,为肾综合征出血热(HFRS)发病机理、疫苗研制及抗病毒免疫反应研究奠定基础。方法　采用Ficoll密度梯度离心法分离HFRS恢复期患者外周血单个核细胞 (PBMC)。用IFN γELISPOT实验和T细胞增殖实验 ,测试 7名患者PBMC对 2 3条NPC 端合成多肽的T细胞应答。结果　IFN γELISPOT实验结果表明 ,2名供体(3、4 )可分别检测到对 5 1、70号 2条多肽特异性T细胞应答。在供体 3,70号肽特异性T细胞频率为4 5SFC 10 6 PBMC ;在供体 4 ,5 1号肽特异性T细胞频率为 82SFC 10 6 PBMC。T细胞增殖实验与ELISPOT结果基本一致 ,但 5 3号肽和 6 4号肽还可分别刺激供体 1和供体 4的T细胞增殖 ,而未能诱导IFN γ分泌。结论　 5 1号和 70号多肽可能是NPC 端较强的T细胞表位。     

Microarray analyses of differentially expressed human genes and biological processes in ECV304 cells infected with rubella virus

Changes in mRNA expression levels of ECV304 cells infected with the wild-type rubella strain were analyzed using a microarray system representing 18,716 human genes. Four hundred eighty-seven genes exhibited differential expression levels; 456 of these genes were up-regulated while 31 genes were down-regulated. We identified 53 biological processes that were significantly relevant to the RV-infection. Among these biological processes, 52 were one-gene processes and one was a process involving five genes: IFNA21 (interferon, alpha 21), interferon stimulated exonuclease gene 20 kDa (ISG20), zinc finger protein 175 (ZNF175), tripartite motif-containing 22 (TRIM22), and MX2 [myxovirus (influenza virus) resistance 2 (mouse)]. Except for ZNF175, gene annotation indicated four of these genes encoded interferon or interferon-induced genes. These results suggest that genes relevant to interferon-regulated pathways may be involved in the pathogenesis of rubella.     

Ⅰ型出血热患者中性粒细胞与淋巴细胞功能变化研究

目的研究肾综合征出血热（ＨＦＲＳ）患者的淋巴细胞和中性粒细胞免疫功能变化及其相互间关系，进一步阐明发病机理。方法采用Ｅａ、Ｅｔ花环形成、ＰＨＡ皮肤试验及中性粒细胞趋化、吞噬、ＮＢＴ还原等多种试验方法同时对３２例由Ｈａｎｔａａｎ病毒引起的姬鼠型ＨＦＲＳ患者的淋巴细胞和中性粒细胞免疫功能变化及其相互间关系进行动态测定。结果ＨＦＲＳ患者早期前五项指标均明显低于正常人（Ｐ＜０．０１）；病程中，淋巴细胞损伤程度较粒细胞更加突出；中性粒细胞ＮＢＴ还原能力在恢复期反降至最低（Ｐ＜０．０５）。此外，患者这两种细胞的免疫功能在恢复期并未能完全达到正常水平。结论ＨＦＲＳ患者淋巴细胞免疫功能损伤程度与病程密切相关，可能是Ｈａｎｔａａｎ病毒直接作用的结果；而中性粒细胞免疫功能变化比较复杂，有待进一步研究。恢复期患者细胞免疫功能未能完全恢复提示临床治疗应予以关注。     

Seroepidemiology of Hantaan virus infection in Taiwan

In order to investigate the infection rate of Hantaan virus in Taiwan, a total of 6,536 human serum samples were collected from residents, selected by stratified random sampling, from 19 townships covering four different ethnic groups: Aborigines, Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese. Serum samples were screened for Hantaan virus antibodies by indirect immunofluorescence. The prototype Hantaan virus (76/118)-infected Vero E6 cells were used as the viral antigen for the antibody detection. Among 6,536 human serum samples, 403 (6.2%) samples had Hantaan virus antibodies. The seropositive rates for males and females were 6.1% and 6.2%, respectively. A higher seropositive rate was found among Aborigines on the Orchid Islets (11.5%) and Fukien Taiwanese on the Penghu Islets (11.6%), while the lowest rate was observed among Hakka Taiwanese in the south of Taiwan (2.5%). In comparing with different ethnic groups, the highest prevalence was found among Fukien Taiwanese (8.1%) and the lowest among Mainland Chinese (4.9%). Among the different geographical areas, the highest positive rate was found in western Taiwan (7.1%) and the lowest in southern Taiwan (5.4%). Hantaan virus antibodies were also detected in 22 of 548 (4.0%) rat serum samples. The highest seropositive rate was found in rat sera collected from the Orchid Islets (21.4%). None of the rat sera collected from Hsinchu, Miaoli, Changhua, Nantu, Yunlin, Chi-ayi, Tainan, and Penghu Counties were positive. Hantaan virus antibodies were found in rats: Rattus rattus (20%), Bandicota indica (9.0%), Rattus norvegicus (8.3%), Bandicota nemorivaga (6.3%), Rattus losea (4.2%), and Apodemus agrarius (1.6%). Hantaan virus antibodies were not detected in rat sera collected from species of Rattus coxinga, Rattus culturatus, Mus musculus, Mus caroli, Suncus murinus, and Apodemus semotus. The results show that the Hantaan or Hantaan-related virus exists and is distributed widely in both human and rats in Taiwan. © 1996 Wiley-Liss, Inc.     

汉滩病毒结构蛋白昆虫细胞融合表达产物的免疫原性评价

目的：研究汉滩病毒（HTNV）囊膜糖蛋白G1与核蛋白（NP）部分片段在昆虫细胞中融合表达产物的免疫学特性，为HTNV基因工程疫苗的研制提供依据。方法：构建含有HTNV G1S0.7嵌合基因的重组杆状病毒Bac-G1S0.7，用其感染的Sf9细胞免疫Balb/c小鼠。用间接免疫荧光法、ELISA法、微量细胞培养中和试验及T淋巴细胞增殖试验对被免疫小鼠的体液免疫及细胞免疫应答效果进行检测。结果：Bac-G1S0.7感染的昆虫细胞免疫小鼠后，测得免疫鼠血清抗HTNV抗体滴度最高达1：3200，含有特异性抗HTNV NP及抗HTNV糖蛋白G1的抗体，被免疫小鼠中有部分产生了较低滴度的中和抗体，免疫鼠脾细胞对HTNVNP或糖蛋白的增殖反应指数均高于阴性对照组。结论：G1S.07嵌合基因的昆虫细胞表达产物具有较好的免疫原性，可刺激机体同时产生针对HTNV NP与囊膜糖蛋白G1的细胞及体液免疫应答。     

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.     

Modulation of apoptosis and immune signaling pathways by the Hantaan virus nucleocapsid protein

Herein, we show a direct relationship between the Hantaan virus (HTNV) nucleocapsid (N) protein and the modulation of apoptosis. We observed an increase in caspase-7 and -8, but not -9 in cells expressing HTNV N protein mutants lacking amino acids 270-330. Similar results were observed for the New World hantavirus, Andes virus. Nuclear factor kappa B (NF-κB) was sequestered in the cytoplasm after tumor necrosis factor receptor (TNFR) stimulation in cells expressing HTNV N protein. Further, TNFR stimulated cells expressing HTNV N protein inhibited caspase activation. In contrast, cells expressing N protein truncations lacking the region from amino acids 270-330 were unable to inhibit nuclear import of NF-κB and the mutants also triggered caspase activity. These results suggest that the HTNV circumvents host antiviral signaling and apoptotic response mediated by the TNFR pathway through host interactions with the N protein.     

Herpes simplex virus 1 induced LOX-1 expression in an endothelial cell line, ECV 304

Infections, such as by Chlamydophilia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori, have been shown to be involved in atherogenesis. Herpes simplex virus I (HSV-1) could infect vascular endothelial cells, and it has been shown that, when endothelial cells were activated with oxidized LDL (oxLDL), a number of cellular events are occurred, leading to endothelial cell dysfunction. Since LOX-1 is a major receptor for oxLDL on endothelial cells and its expression was increased in atherosclerosis, we investigated whether HSV1 infection can lead to the increase expression of LOX-1 in endothelial cells. LOX-1 mRNA expression determined by RT-PCR and LOX-1 promoter activity measured by luciferase assay were increased in endothelial cells following HSV-1 infection. This suggests that one of the mechanisms by which HSV-1 is involved in atherogenesis maybe the enhanced uptake of oxLDL via the increased expression of LOX-1 in endothelial cells.     

氧化高密度脂蛋白通过MAPKs信号转导途径诱导ECV304细胞表达组织因子

目的： 探讨氧化高密度脂蛋白(oxHDL)对人脐静脉内皮细胞株ECV304细胞表达组织因子(TF)的影响及其机制。方法：实验分为阴性对照组、阳性对照组（加组胺）、HDL、oxHDL 4组, ECV304细胞经不同浓度oxHDL、HDL（10-120 mg/L）和组胺(1×10-9-1×10-4 mol/L)处理不同时间(2-8 h)后,采用实时定量PCR (RQ-PCR)和Western bloting方法检测TF表达。同时,分别应用SP600125［c-Jun氨基端激酶(JNK) 特异性抑制剂］、SB203580［p38丝裂素活化蛋白激酶(p38MAPK) 特异性抑制剂］、 PD98059［细胞外信号调节激酶(ERK1/2) 特异性抑制剂］,观察其对oxHDL和组胺诱导的ECV304细胞表达TF的影响。 结果：正常ECV304细胞未检测到TF,经组胺处理1 h后TF mRNA表达明显增加,在1×10-5 mol/L时最高,约为1×10-9 mol/L时的4.8倍。oxHDL以时间和浓度依赖性方式诱导ECV304细胞TF mRNA表达,在oxHDL 20 mg/L时最高,为10 mg/L时的1.8倍,在第6 h时TF mRNA表达水平为第2 h的5.3倍（P<0.05）,并在20 mg/L时显著刺激ECV304细胞TF蛋白表达,为40 mg/L时的1.5倍,而HDL没有此效应。 MAPKs（JNK、p38 MAPK、ERK1/2）抑制剂可部分解除oxHDL诱导ECV304细胞TF表达的效应,TF分别下调表达至原来的19.0%、5.0%和13.0%（P<0.05)。结论：oxHDL能以浓度和时间依赖性方式诱导ECV304细胞产生TF,其机制可能通过JNK、p38 MAPK和ERK1/2所介导。     

免疫小鼠巨噬细胞的抗汉坦病毒感染作用

用四甲基偶氮唑蓝比色法测定了汉坦病毒对小鼠腹腔巨噬细胞细胞活力的抑制作用，发现其抑制率的高低与感染时间及细胞内的病毒抗原量具有相关性。重组汉坦病毒核壳蛋白和重组糖蛋白免疫小鼠巨噬细胞再接种ＨＴＮＶ后细胞活力与未感染巨噬细胞相比，未见明显降低，细胞内病毒抗原阴性，表明免疫小鼠的巨噬细胞具有抵抗汉坦病毒感染的作用。