PPARγ激动剂抗呼吸道合胞病毒感染作用的体外研究

目的 探讨过氧化物酶体增殖物活化受体γ(PPARγ)激动剂的体外抗呼吸道合胞病毒(RSv)感染作用.方法 以细胞存活率和病毒抑制率为指标,应用细胞病变效应(CPE)法,观察不同浓度PPARγ激动剂对人肺腺癌(A549)细胞的CPE及RSV感染后CPE的抑制作用.采用四甲基偶氮唑蓝(MTT)比色法检测PPARγ激动剂对A549细胞的毒性作用和RSV感染后细胞存活率和病毒抑制率的影响.结果 5～25μ mol/L15-脱氧前列腺素J2( 15 d-PGJ2)和10～50μmol/L罗格列酮干预的A549细胞未显示明显CPE,MTT比色法也显示以上浓度范围15 d-PCJ2和罗格列酮的A值明显高于RSV感染组(P＜0.01),但两种药物相比差异无统计学意义.15d-PGJ2和罗格列酮最适浓度分别为5μmol/L和10 μmol/L.结论 PPARγ激动剂既可减轻RSV感染后A549细胞的CPE,又可提高细胞存活率,具有体外抗RSV感染作用.     

乌鲁木齐地区冬春季小儿呼吸道感染者中呼吸道合胞病毒感染的研究

目的 了解乌鲁木齐地区冬春季节呼吸道感染患儿中呼吸道合胞病毒(RSV)的感染状况以及分子流行病学的基本情况.方法 研究对象为2006年11月-2007年4月于新疆维吾尔自治区人民医院儿科住院以及部分于门诊就诊,明确诊断为急性呼吸道感染的患儿,采集咽拭子280份,呼吸道分泌物112份.对全部标本采用反转录聚合酶链反应(RT-PCR)方法进行RSV及其亚型检测.随机取5份阳性标本测序,进行序列比较及同源性分析.结果 392份样本中共检出RSV阳性68份,阳性率17.3%(68/392),其中A基因型64份,占93.3%(64/68),B基因型4份,占6.7%(4/68).5株测序结果提示当地RSV与其他国家及地区代表株之间的同源性为63.1%～99.4%.进化树进一步显示存在A、B两个亚型.结论 RSV是引起2006-2007年冬春季该医院儿童急性呼吸道感染的重要病原体,以A亚型为主要流行株.

Abstract：

Objective To research the infections of respiratory syneytial virus(RSV)in children with respiratory tract inflammation and define its molecular epidemic features in Urumchi.Methods SamDles were collected from November 2006 to April 2007 in the People's General Hospital of Xinjiang Uygur Autonomous Region,including 112 respiratory secretions and 280 nasopharyngeal swabs. RSV and its subgroups were detected by nested PCR.The five positive amplicons selected randomly from all positive samples were sequenced and compared with other RSV in GenBank by BLAST and DNAStar.Results of all 392specimens.68 RSV G gene segments were tested.Among them,RSV lineage A occupied 93.3%,while B occuDied 6.7%.The identities between them were 63.1%-99.4%.Phylogenetic analysis defined that they belonged to two different clusters.Conclusion RSV was one of the important viruses leading to children's respiratory tract infections in the People's General Hospital of Xinjiang Uygur Autonomous Region during winter and spring from 2006 to 2007.RSV subtype A was the prevalent genotype in the hospital dunng this epidemics.     

金银花体外抗呼吸道合胞病毒的作用研究

目的了解金银花体外抑制呼吸道合胞病毒的效果和强度。方法采用细胞病变抑制实验．噻唑蓝（MTT）比色法检测细胞活性,观察金银花在人宫颈癌传代细胞（Hela）中对人呼吸道合胞病毒3型的抑制作用,以治疗指数（TT）为评价指标。结果金银花对Hela细胞半数中毒浓度（TC50）为5mg／ml,最大无毒浓度（TC50）为3．6mg／ml;对呼吸道合胞病毒有直接灭活作用,其半数抑制率（IC50）为0．16mg／ml,TI为31．2;在吸附阶段也有作用,其IC50为0．48mg／ml,TI为10．5;同时金银花有抑制呼吸道合胞病毒生物合成作用,其IC50为1．0mg／ml,TI为5;金银花不能阻止呼吸道合胞病毒侵入细胞。结论金银花在体外主要通过直接灭活、阻止病毒吸附和抑制生物合成3种方式发挥抗呼吸道合胞病毒作用。     

呼吸道合胞病毒感染的研究进展

呼吸道合胞病毒是在世界范围内引起婴幼儿呼吸道感染的最常见病原微生物,它不仅可以引起呼吸系统的一些常见症状和体征,而且在肺外器官如中枢神经系统、心血管系统、内分泌系统等各器官也可引起一些尚未被人们普遍认识的肺外表现,而这些临床特点的重要性也愈来愈受到人们的重视.     

呼吸道合胞病毒感染患儿的血锌及免疫功能改变

<正> 呼吸道合胞病毒(RSV)是引起婴幼儿呼吸道感染的主要病原之一。为观察血锌及T细胞亚群与RSV感染的关系,本文对42例患儿血锌、细胞及体液免疫作了检测,以了解其变化及相互关系。     

呼吸道合胞病毒感染与细胞凋亡的研究进展

呼吸道合胞病毒感染和细胞凋亡的关系是错综复杂的,既可以表现为抗凋亡作用,以利于病毒在感染细胞内复制;也可以表现为促凋亡作用,此可能是机体对病毒感染的防御反应,亦可能是病毒感染导致宿主组织细胞严重损伤的重要机制.研究呼吸道合胞病毒感染与细胞凋亡关系及凋亡的可能机制,将有助于进一步认识呼吸道合胞病毒感染发生、发展及转归机制,为RSV感染的防治提供一些新的思路.     

婴幼儿呼吸道合胞病毒感染分子流行病学研究

目的　探讨婴幼儿呼吸道合胞病毒(RSV)感染的分子流行病学情况。方法　采用随机扩增多态DNA(RAPD)技术,对长春市儿童医院1992～1994年分离并鉴定的96株RSV和一株标准RSV进行RAPD分析。结果　所有RSV毒株都有扩增带,共有四种带型。不同疾病来源的RSV基因型不同。来源毛细支气管炎的RSV有8714%为R1型。结论　RAPD技术能从分子水平了解婴幼儿RSV感染情况;R1型RSV可能为引起婴幼儿毛细支气管炎的主要病原。     

脱氧核酶抗呼吸道合胞病毒感染不同免疫功能小鼠的研究

目的 研究脱氧核酶(DZ)抗不同免疫功能小鼠呼吸道合胞病毒(RSV)感染的作用.方法 RSV感染BALB/c鼠和裸鼠滴鼻给予DZ,空斑形成试验检测肺组织病毒滴度,RT-PCR检测病毒mRNA表达、支气管肺泡灌洗液白细胞计数,ELISA检测TNF-o、IL-12、IFN-γ和IL-10水平,肺组织病理学分析炎症情况.结果 DZ治疗组BALB/c鼠和裸鼠肺组织病毒滴度比感染对照组下降(P＜0.05),裸鼠下降更明显(P＜0.01).0.2 mg、0.4 mg和0.8 mg DZ分别降低感染BALB/c鼠30.51%、47.38%(P＜0.05)、53.97%(P＜0.01)和感染裸鼠36.59%(P＜0.05)、48.72%、59.78%(P＜0.01)病毒mRNA表达.0.4 mg DZ治疗降低感染BALB/c鼠和裸鼠支气管肺泡灌洗液中白细胞总数,改善肺组织病理学损伤(P＜0.05),降低感染裸鼠气道局部TNF-α、IL-12和IFN-γ分泌(P＜0.05).结论 DZ在不同免疫功能小鼠体内有效抑制RSV复制,减轻气道炎症,对裸鼠的保护作用更突出,是有效的抗RSV制剂.     

呼吸道合胞病毒感染人肺上皮细胞活化Toll样受体3介导的抗病毒作用研究

目的 探讨呼吸道合胞病毒( RSV)感染人肺上皮A549细胞后,Toll样受体3(TLR3)的水平变化及其产生的Ⅰ型干扰素的抗病毒作用.方法 RSV感染体外培养的人肺上皮A549细胞,并给予TLR3特异性抗体处理,分别感染4、8、12、16、24h后收集各组细胞.未感染病毒的细胞作为对照组.RT-PCR法检测TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达水平变化.结果 RSV感染A549细胞后,TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达量均升高且有时间依赖性,TLR3 mRNA在24h表达量是基础表达量的5倍,IFN-α、IFN-β mRNA在24 h表达量是基础表达量的4倍多,RSVF蛋白的mRNA表达量近1.7倍.TLR3抗体预先处理以抑制TLR3受体,再行RSV感染,IFN-α和IFN-β mRNA表达量虽然升高,但较感染组均有所下降,mRNA表达在12 h后显著降低,且IFN-ββ的mRNA表达量下调更明显.但RSV F基因的mRNA表达在12 h、24 h升高有显著性差异.结论 RSV感染A549细胞后可上调TLR3表达,其活化细胞介导产生的Ⅰ型干扰素起抗病毒作用,在一定程度上可抑制病毒的增殖水平.     

The immunobiology of respiratory syncytial virus infection

There is increasing evidence that young children with severe respiratory syncytial virus (RSV)-induced bronchiolitis are at high risk of developing allergy and asthma during their later life. The determinants for this association are not well understood. Current studies suggest that both genetic backgrounds and unique characteristics of the virus play critical roles in determining the type of immune responses to RSV infection, leading to altered regulation of airway tone associated with wheezing. In susceptible subjects, RSV may either enhance the Th2 immune response or decrease the Th1 immune response. This altered Th1/Th2 cytokine response associated with RSV infection is not commonly observed among other RNA viruses, suggesting that RSV may have unique characteristics. Multiple clinical studies support the link between severe RSV bronchiolitis and the subsequent development of allergy and asthma. This link will be further tested by the ongoing large studies on the effect of early RSV intervention on the development of allergy. The administration of palivizumab, an anti-RSV monoclonal antibody, seems to be helpful for RSV prevention and treatment at early stage. There are no effective RSV vaccines available, and this is, at least in part, because of the poorly understood immunology and pathogenesis of RSV disease. The use of experimental animal models has led to a better, but not sufficient, understanding of the immunologic basis of RSV-induced disease, particularly asthma. Further studies on the immunopathology of RSV infection with animal models, including the nonhuman primate models, may help develop effective RSV vaccines.     

Role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro

RSV infection in airway epithelial cells (EC) results in production of the chemokines RANTES and MIP1alpha and the leukocyte differentiation factor GM-CSF. The chemokines attract monocytes and eosinophils to the site of infection, where GM-CSF may influence their function and differentiation. In turn, these inflammatory cells may limit the progression of RSV infection, as well as initiate immune responses. In the present study, the effect of monocytes and eosinophils on viral replication and infection-dependent release of EC-derived cytokines was investigated. The modulation of immune cell costimulatory molecules, CD80, CD86, CD40, and HLA-DR, and the release of the CD4(+) T cell chemoattractant IL-16 were also investigated. Employing immunofluorescence techniques, monocytes and eosinophils in cocultures with infected EC were found to inhibit the spread of RSV to uninfected cells. Monocytes also had a significant effect on replication of RSV. Monocytes phagocytized the virus, while eosinophils inhibited reinfection mainly by extracellular means. The release of G-CSF and GM-CSF in the infected cultures was not significantly affected by either monocytes or eosinophils, while RANTES release was significantly decreased. The expression of CD40, CD80, CD86, and HLA-DR on monocytes, but not on eosinophils, increased in an RSV-dose-dependent manner. IL-16 release was not induced in RSV-infected EC, but was significantly increased in coculture with monocytes. These results suggest that both monocytes and eosinophils attracted to the site of RSV infection play an important role in confining infection, while RSV-exposed monocytes may be involved in promoting/polarizing immune responses to RSV.     

呼吸道合胞病毒下呼吸道感染对机体细胞免疫的影响

为研究呼吸道合胞病毒（ＲＳＶ）急性下呼吸道感染（ＡＬＲＩ）的细胞免疫变化，对２５例病儿外周血白细胞介素２（ＩＬ－２）和可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）水平、Ｔ细胞白细胞介素２受体（ＩＬ－２Ｒ）表达率和Ｔ细胞亚群百分率进行检测。结果显示，急性期病儿外周血ＩＬ－２水平明显低于恢复期和正常对照组，Ｔ细胞ＩＬ－２Ｒ表达率亦明显降低，而ｓＩＬ－２Ｒ水平却显著增高。急性期病儿ＩＬ－２水平与Ｔ细胞ＩＬ－２Ｒ表达率和ＣＤ＋４细胞百分率呈正相关，与ｓＩＬ－２Ｒ水平和ＣＤ＋８细胞百分率呈负相关；ｓＩＬ－２Ｒ水平与Ｔ细胞ＩＬ－２Ｒ表达率呈负相关，与临床严重程度呈正相关。上述各项免疫指标异常均提示ＲＳＶ感染时机体存在细胞免疫功能紊乱。     

蛭丹化瘀口服液体外抑制RSV作用机制的研究

目的 研究蛭丹化瘀口服液体外抗呼吸道合胞病毒作用环节.方法 观察该药在不同浓度和不同的作用环节下RSV对Hep-2细胞的致病作用.结果 蛭丹化瘀口服液对细胞无毒浓度为5.5 mg/ml,并在此浓度中未能阻断RSV对细胞的吸附,细胞产生了完全病变.蛭丹化瘀口服液在2.75～5.50 mg/ml浓度中,药物直接灭活病毒组和先感染细胞再加入药物的治疗组均未产生细胞病变.结论 蛭丹化瘀口服液体外实验无预防RSV感染作用,有直接灭活RSV作用,并对进入细胞内的RSV有抑制作用.     

Respiratory syncytial virus infection in immunocompetent and immunocompromised murine

The purpose of this study is to distinguish respiratory syncytial virus ( RSV) infection and immunology between immunocompetent and immunocompromised murine and to explore immune mechanism of RSV infection. At various time points after RSV infection of BALB/c mice and nude mice, pulmonary viral titers were assayed, RSV antigen was tested by direct immune-fluorescent assay and immu nohistochemistry. Pulmonary mRNA expressions of Toll like receptor (TLR)2 and TLR4 were assayed by RT-PCR. CD4 cells and CD8 cells in peripheral blood were examined by flow cytometry and plasma total IgE was assayed by ELISA. Leukocytes in bronchoalveolar lavage fluid (BALF) and pulmonary histology were identified to reflect airway inflammation. It was found that RSV titers of both mice peaked on the 3rd day post infection with a much higher level of viral titer in nude mice than in BALB/c mice and a longer viral duration in nude mice (over 9 days post infection) than in BALB/c mice (6 days post infection). RSV infection induced higher viral antigen expression in nude mice (0.267±0.045) than in BALB/c mice (0.168±0.031). RSV infection enhanced pulmonary TLR4 expression of BALB/c mice (51.96%±11.34%) and nude mice (48.96%±12.35%) compared with each control (34.04%±10.06% and 32.37%±9.87% respectively). CD4 peripheral blood cells increased in RSV infected BALB/c mice (66.51%±2.09%) compared with the control BALB/c mice (51.63%±5.90%), and CD4 cells and CD8 cells were deficient in nude mice. RSV infection increased plasma total IgE in both mice, and BALB/c mice had a larger amount of IgE on the 7th day post infection (9.02 ng/ml±2.90 ng/ml) and on the 14th day post infection (12.76 ng/ml±4.15 ng/ml) than corresponding nude mice (3.72 ng/ml±1.06 ng/ml and 7.62 ng/ml±3.08 ng/ml respectively on the 7th and 14th day post infection). RSV infected nude mice had more severe airway inflammation than infected BALB/c mice. It is concluded that BALB/c mice and nude mice presented similar RSV infectious characteristics. However, infection of nude mice showed higher viral titer with longer duration and more severe airway inflammation, lower level of plasm total IgE and CD4 peripheral blood cells, but the similar pulmonary TLR4 expression with BALB/c mice.     

呼吸道合胞病毒单克隆抗体逃逸株体外适合度变化

目的 Palivizumab(PZ)是针对呼吸道合胞病毒(respiratory syncytial virus,RSV)F蛋白的人源化单克隆抗体(ScAb).本研究观察PZ逃逸株体外适合度变化规律.方法 采用竞争复制法测定两株Pz抵抗株(F212和MP4)的体外适合度.将等量F212和母株A2混合后在HEp-2细胞中连续转代,在不同时间点采用差异噬斑法、核酸序列图谱分析法和限制性片段多态性法分析混合病毒群体中F212和A2的比例.由于MP4在细胞培养和棉鼠肺内复制水平与A2相似,故将A2和MP4按照1:1、10:1、100:1和1000:1的比例混合并在HEp-2细胞中竞争复制,不同时点采用差异噬斑法和核酸序列图谱分析法分析混合病毒群体中MP4和A2比例.结果 等比例混合的F212/A2混合群体在传代至10代时,F212已基本消失,传至35代时仍无F212信号,提示其适合度低于A2.A2/MP4混合病毒群体中,除按1000:1比例预混外,其余所有比例预混的病毒中MP4均成为优势株,因而提示MP4的适合度高于A2.结论 首次发现RSV PZ逃逸株适合度可超过原型株A2,了解适合度增加或降低的机制对减毒活疫苗的研究有理论指导意义.