人巨细胞病毒感染巨核祖细胞及反义寡核苷酸抗病毒感染作用的研究

目的探讨人巨细胞病毒 (HCMV)对人巨核细胞及其前体细胞的抑制作用及其机制 ,并观察HCMV反义寡核苷酸 (ASON)的抗病毒感染作用。方法在半固体培养基上定向诱导CD34 细胞向巨核细胞分化。用HCMVAD16 9病毒株感染培养的巨核细胞和经ASON预处理的CD34 细胞 ,观察CFU MK形成率的变化 ,分别用PCR、RT PCR法检测CFU MK细胞中HCMV即刻早期蛋白 (IEP)基因DNA和mRNA、即刻早期基因 (UL36 )mRNA表达。用MTT法测定ASON的细胞毒性。结果CD34 细胞在半固体培养基中定向分化为巨核祖细胞。HCMVAD16 9株能明显抑制巨核细胞的体外增殖 ,与灭活病毒组比较 ,三个不同病毒滴度感染组的CFU MK分别减少了 2 1.6 %、33.8%、4 6 .3% ,显示抑制程度与病毒感染滴度呈剂量依赖关系 ;HCMV感染的CFU MK细胞中可检出HCMVIEP基因DNA、IEP基因mRNA和UL36mRNA。 0 .0 8μmol/L的UL36ASON (UL36Anti)能使HCMV感染细胞CFU MK形成率恢复正常 ,与 10 0 0 0TCID50 HCMV感染组比较 (分别为 5 8.2 4± 7.4 2和 31.17± 4 .4 5 ) ,差异有显著性 (P<0 .0 5 ) ,RT PCR未能检出UL36mRNA。而改变UL36Anti中 1个碱基的ASON(MM1) ,浓度只有达到 0 .4 0 μmol/L才能对抗HCMV的作用。MTT结果表明UL36Anti显著影响细胞生长的浓度为 90 .0 0 μmol/L。结     

人巨细胞病毒蛋白抗原的研究进展

人巨细胞病毒(HCMV)属于疱疹病毒β亚科,人群感染率高达80%-90%,大多数感染的初期是一个潜伏状态或低量的慢性、非典型的隐性感染[1],但在感染孕妇、婴幼儿以及器官移植受体和免疫抑制个体时,可通过病毒的潜伏-激活机制而发病,导致严重的感染或相关的并发症。在HCMV感染的细胞周期中,分为即刻早期(IE)、早期(E)、晚期(L)三个时相,其间病毒基因所表达的IEI(UL123)I、E2(UL122)、pp52(UL44)、pp65(UL83)和pp150(UL32)几种重要蛋白抗原,对病毒的复制及细胞周期的进程有着十分重要的影响,现作一综述。1 IE1和IE2 IE1是由UL123编码…     

人类巨细胞病毒的变异性分析

目的探讨人巨细胞病毒(HCMV)基因组的4个片段在人群和单个病人病程中变异性的临床意义.方法通过聚合酶链反应(PCR)对编码糖蛋白B(UL55)、编码DNA单链结合蛋白(UL57)、编码主要衣壳蛋白(UL86)、编码IE抗原(UL122)4个基因片段进行扩增后,用限制性内切酶对扩增片段进行酶切验证,再使用HCMV的标准株AD169进行试验条件的优化,然后对200份临床可疑HCMV感染血标本进行检测.结果 200份血清标本中UL55基因PCR阳性率12.5%,酶切阳性率10.0%,与其他3个基因片段比较差异有显著性(P<0.05);在3例HCMV感染持续阳性的病人血清系列标本中UL55基因存在阳性转阴性现象.结论对HCMV进行PCR检测,应选择变异性较小的基因片段;HCMV通过UL55基因的变异可逃避人体免疫系统对其的攻击.     

活动性人巨细胞病毒感染的病毒基因表达特点

目的:探讨活动性人巨细胞病毒(HCMV)感染的病毒基因表达特点.方法:建立持续稳定感染(A组)及活动感染(B组)两种细胞感染状态,FQ-PCR和RT-PCR法监测HCMV DNA复制和MCP mRNA转录,免疫组化法联合计算机病理图文分析系统检测pUL44表达水平,光镜观察细胞病变、电镜检查细胞超微结构改变.结果:B组HCMV DNA负荷量(5.61～9.04 lgGE/106HEL)明显高于A组(4.32～5.91 lgGE/106HEL,P<0.05或P<0.01),pUL44也持续升高表达(area:339.6～1 431.9μm2,IA:56.4～319.8,P<0.01),MCP mRNA水平逐渐升高.A组pUL44少量、随机表达(area:219.2～296.7μm2,IA:31.0～58.3),未检到MCP mRNA,亦未发现细胞病变,B组出现进行性加重的细胞病变和细胞超微结构破坏.结论:HCMV DNA负荷量持续升高及MCP mRNA转录是活动性HCMV感染的重要标志.     

人巨细胞病毒UL123基因全长及其外显子4的克隆与表达

【目的】克隆人巨细胞病毒（HCMV）UL123基因cDNA全长（iel）及其外显子4（iel-exon4），构建原核表达重组质粒，诱导立即早期蛋白1（IE1）及其C-端86-491氨基酸（IE1-C86-491）表达，并鉴定表达产物。【方法】分别用RT-PCR和PCR法将iel和iel-exon 4从感染了HCMV的人胚肺成纤维细胞（HEL）中扩增出来，再分别克隆入原核表达载体pTWIN1上intein2的N端，转化大肠杆菌，经PCR、限制性酶切和测序鉴定阳性重组子，在低温和低IPTG浓度下诱导重组子表达可溶性重组融合蛋白rIE1／intein2和rIE1-C86-491／intein2，用SDS-PAGE和Western Blotting对表达产物进行鉴定。【结果】经PCR、限制性酶切和测序鉴定重组质粒pTWIN1／iel和pTWIN1／iel-exon4构建成功，sDS-PAGE和Western Blotting分析表明融合蛋白在大肠杆菌中表达。【结论】成功克隆并表达了iel和iel-exon4。     

白细胞介素13对HEL细胞分化和转录因子c-fos表达的影响

目的研究白细胞介素13（IL-13）对红白血病细胞系HEL细胞分化作用及对HEL细胞转录因子c-fos表达的影响。方法用RT—PCR方法检测HEL细胞IL-13受体仪1、GPⅡb、vWF和c-fos基因mRNA表达，用Western blot和流式细胞术分别检测IL-13受体α1、c-fos、GPⅡb和vwF蛋白水平的表达。结果HEL细胞表达IL-13受体α1，用IL-13（100ng／ml）作用于HEL细胞，GPⅡb和vWF基因mRNA表达上调，实验组和对照组GPⅡb／β-actin吸光度比值分别为2．912，1．303，实验组为对照组的2．23倍（P〈0．05）；实验组和对照组vWV／f3一actin吸光度比值分别为0．506，0．217，实验组为对照组的2．33倍（P〈0．05）。流式细胞术分析结果显示，实验组GPⅡb和vWF蛋白表达明显高于对照组。IL-13作用于HEL细胞30min，c—fos基因mRNA表达水平达高峰，IL-13作用HEL细胞60min，c—fos蛋白表达达高峰，30min组、60min组c-fos/β-actin吸光度比值高于其它各组（P〈0．05）。结论IL-13可促进HEL细胞分化，并上调c-fos表达。     

高三尖杉酯碱联合AG490对HEL细胞JAK2-STAT5相关信号通路的影响

本研究旨在探讨高三尖杉酯碱( homoharringtonine,HHT)联合AG490对HEL细胞JAK2-STAT5信号通路的影响及其机制,为临床应用新方案治疗慢性骨髓增殖性肿瘤( MPN)提供理论依据.以20 ng/ml的HHT,100μmol/L AG490,20 ng/ml HHT+ 100 μmol/L AG490处理HEL细胞24、48、72小时以后,用MTT法和流式细胞术检测细胞生长抑制率和凋亡率,药物处理细胞24小时后用WB法检测JAK2突变激活的信号蛋白P-JAK2,P-STAT5以及BCL-xL表达的变化.结果表明,HHT以及AG490作用HEL24小时均能抑制其增长,Annexin V-PI双染流式细胞图显示均能明显诱导HEL早期凋亡,HHT更为明显;免疫电泳分析显示,P-JAK2和P-STAT5表达下调,而JAK2和STATS总蛋白水平稳定.结论:HHT联合AG490能明显抑制HEL细胞增殖并诱导凋亡,两者具有协同作用,其作用机制是HHT作为一种广谱的蛋白酪氨酸激酶抑制剂,协同AG490抑制JAK2突变引起的信号蛋白的酪氨酸位点的磷酸化,从而下调STAT5反应性基因的转录.     

靶向UL5小干扰RNA对Ⅱ型单纯疱疹病毒的抑制

背景:解旋酶-引发酶复合体是Ⅱ型单纯疱疹病毒进行复制的必需基因,UL5基因为Ⅱ型单纯疱疹病毒解旋酶-引发酶复合体的组成单位之一。目的:应用RNA干扰技术分析特异性小干扰RNA对Ⅱ型单纯疱疹病毒UL5基因的干扰作用。方法:以Ⅱ型单纯疱疹病毒UL5基因为靶目标,设计、合成5对特异性小干扰RNA。通过LipofeCtamine 2000脂质体将特异性小干扰RNA转染HEK293细胞。48h后行荧光定量RT-PCR检测UL5基因转录水平,终点滴定法测定干扰后病毒滴度,观察其干扰效果。结果与结论:小干扰RNA成功转染入细胞内。荧光定量RT-PCR结果显示,siRNA722、siRNA2394、siRNA2513和siRNA2627均可不同程度降低靶mRNA表达,病毒滴度检测结果显示siRNA722、siRNA2394、siRNA2513和siRNA2627均可不同程度降低上清液中病毒感染滴度,阴性对照组和siRNA374对病毒感染滴度无影响。针对UL5基因的有效小干扰RNA可以特异性降低Ⅱ型单纯疱疹病毒的复制水平。     

短发夹RNA表达载体对2型单纯疱疹病毒UL54基因的干扰效应

目的研究短发夹核糖核酸(shRNA)表达载体对2型单纯疱疹病毒(HSV-2)UL54基因的干扰效应。方法针对HSV-2 UL54基因,构建5个靶向HSV-2 UL54基因的短发夹RNA重组表达载体(shRNA1081、shRNA1092、shRNA1276、shRNA1407、shRNA1508),同时设计不针对任何基因的序列为阴性对照组(阴性对照组)及不加任何重组表达载体的空白对照(空白对照组)。重组表达载体通过脂质体转染人胚胎肾细胞(HEK293)后接种HSV-2,48 h后利用荧光定量RT-PCR检测各组细胞UL54 mRNA的表达水平,终点滴定法测定HSV-2子代病毒滴度。结果荧光定量RT-PCR结果示,与空白对照组相比,shRNA1081、shRNA1407和shRNA1508能够降低HSV-2 UL54 mRNA的表达水平(P均<0.01);对UL54基因的抑制率在阴性对照组(加shRNA NC)、shRNA1081组、shRNA1407组、shRNA1508组分别为7%、69%、56%及55%,以shRNA1081组为最高。终点滴定法结果示,与空白对照组比较,shRNA 1081组、shRNA 1407组、shRNA 1508组病毒滴度不同程度下降(P均<0.01)。结论成功构建UL54 shRNA重组表达载体,shRNA1081、shRNA1407、shRNA1508能在体外细胞水平上不同程度地干扰HSV-2 UL54基因表达,从而抑制HSV-2在HEK293细胞中的复制。     

直接接触骨髓基质HS-5细胞可降低HEL细胞对药物的敏感性

本研究探讨与骨髓基质HS-5细胞直接接触对HEL白血病细胞耐药性的诱发作用及其机制。采用人急性红白血病HEL细胞株与HS-5细胞共培养模式模拟体内白血病细胞及其周围的造血微环境,应用CCK-8法检测HEL细胞对Ara-C,DNR,VP16,MTX等化疗药物的敏感性,流式细胞术检测HEL细胞周期时相分布,实时RT-PCR方法检测p19、p21、p27、MDR1、ABCG2、Bcl-2等基因的mRNA表达,Western blot方法检测p-AktSer473、p-GSK3βSer9、p-STAT3Tyr705、Bcl-2、cleaved-Notch1V1754及Hes1等蛋白表达。结果表明,与HS-5细胞共培养后的HEL细胞对4种化疗药物的敏感性均显著降低。在共培养24 h时,HEL细胞阻滞于G0/G1期,G0/G1期的细胞比例明显增多。PCR结果显示,p21基因的转录水平随着共培养时间的延长逐渐上调;p19基因的mRNA水平在12 h时明显下调,随后又恢复;其它基因的改变均无统计学意义。Western blot结果表明,共培养后HEL细胞的p-AktSer473、p-GSK3βSer9、cleaved-Notch1V1754蛋白及Bcl-2蛋白表达上调,Hes1蛋白表达下调,p-STAT3Tyr705表达无明显变化。结论:直接接触骨髓基质HS-5细胞可增强HEL细胞对化疗药物的抵抗作用。     

Sequence variability of human cytomegalovirus UL146 and UL147 genes in low-passage clinical isolates

OBJECTIVES: Human cytomegalovirus (HCMV) infects a number of organs and cell types in vivo. The different symptoms and tissue tropisms of HCMV infection perhaps result from the genetic polymorphism. A new region of DNA containing at least 19 open reading frames (ORFs - denoted UL133-151) was found in the low-passage HCMV clinical strain Toledo and several other low-passage clinical isolates, but not present in the HCMV laboratory strain AD169. Two of these genes, UL146 and UL147, encode proteins with sequence characteristics of CXC (alpha) chemokines, suggesting that they might influence the behavior of neutrophils during infection. This research was to study the sequence variability of UL146 and UL147 ORFs in HCMV clinical isolates and examine the possible associations between gene variability and the outcome of HCMV infection. METHODS: UL146 and UL147 genes from strains obtained from suspected congenitally HCMV-infected infants were PCR amplified and sequenced. RESULTS: High variability was found in UL146 and UL147 gene among HCMV clinical strains. However, the alpha chemokine motif in UL146 and UL147 genes was conserved in almost all sequences. According to the phylogenetic analysis, all sequences of UL146 in clinical isolates could be divided into three groups. All strains from congenital megacolon infants existed in G2A only, and all from asymptomatic infants existed in G2B peculiarly. CONCLUSIONS: Sequence variability among HCMV clinical strains may affect the ability of UL146 and UL147 to attract human neutrophils and influence viral dissemination. No obvious linkage was observed between UL146 polymorphisms and outcome of suspected congenital HCMV infection.     

人巨细胞病毒对更昔洛韦耐受性的基因型鉴别方法的建立和应用

目的建立实验室检测人巨细胞病毒（HCMV）更昔洛韦（GCV）耐药的基因型分析的常规方法。方法采用巢式聚合酶链反应（PCR）直接扩增33例HCMV感染患者的外周血白细胞及前期实验同步获得的33例临床分离毒株的HCMVUL97基因片段,对扩增产物进行测序,与HCMV标准毒株AD169序列比对,参照国外报道的耐药相关的热点突变,判定各临床分离毒株的耐药基因型。结果除有1株为HCMVUL97呈M460V突变型外,余32株同标准株AD169的序列一致。同一患者的分离毒株与外周血白细胞中测得HCMVUL97基因序列完全一致。结论建立的实验室检测HCMV耐药的基因型分析完全可以替代传统的耐药表型分析,前者无需分离活病毒,不仅需时短,而且相对安全,其技术要点更适宜临床推广应用。     

巨细胞病毒即刻早期基因mRNA检测方法的建立

目的建立快速简便的逆转录聚合酶链反应（ＲＴＰＣＲ）方法检测人巨细胞病毒（ＨＣＭＶ）即刻早期（ＩＥ）基因ｍＲＮＡ。方法设计合成跨越ＨＣＭＶＩＥ基因内含子区的一对引物，分别用ＲＴＰＣＲ和ＰＣＲ技术扩增ＨＣＭＶｍＲＮＡ和ＤＮＡ，用Ｓｏｕｔｈｅｒｎ杂交鉴定阳性产物。结果ＨＣＭＶＩＥ基因ｍＲＮＡ表达在感染后６小时即可出现，并持续到感染后至少９６小时，ＲＴＰＣＲ检测的最低敏感性为１００ｆｇ总ＲＮＡ，其他病毒和细胞ＲＮＡ不能被扩增。结论ＲＴＰＣＲ技术检测ＨＣＭＶｍＲＮＡ敏感、特异，有望应用于临床作为ＨＣＭＶ活动性感染的早期诊断方法     

Novel non-nucleoside inhibitors of cytomegaloviruses (BAY 38-4766): in vitro and in vivo antiviral activity and mechanism of action.

For two decades it has been impossible to develop drugs with novel mechanisms of action against herpesviruses, and treatment has been confined largely to the use of inhibitors of viral DNA polymerase. As a representative of a novel inhibitory approach, the non-nucleosidic BAY 38-4766 was identified as a highly selective inhibitor of human cytomegalovirus (HCMV). The compound selectively inhibits not only HCMV strains, including ganciclovir-resistant, ganciclovir/foscarnet and ganciclovir/cidofovir double-resistant clinical isolates, but also a number of monkey and rodent cytomegaloviruses. In a murine cytomegalovirus (MCMV) pathogenicity model in mice, antiviral efficacy and excellent tolerability were demonstrated. BAY 38-4766-resistant HCMV and MCMV strains are not cross-resistant to the nucleoside analogues ganciclovir and cidofovir or the pyrophosphate analogue foscarnet, indicating a different mode of action. Mechanistic studies demonstrated that the high selectivity of this drug class is most likely due to the inhibition of a late stage of the viral replication cycle. Sequence analyses of resistant HCMV and MCMV strains revealed mutations in UL89 and UL104, proteins known to be involved in viral DNA cleavage and packaging. Consequently, the drug is highly specific for the viral as opposed to cellular functions, since UL89 is related to a bacteriophage terminase and no human equivalent exists. In addition, because some of the genes of the viral DNA cleavage and packaging complex are highly conserved among herpesviruses, development of broad-spectrum agents covering additional human herpesviruses might be possible using this approach.     

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.     

Inhibition of human cytomegalovirus by trifluorothymidine.

The antiviral activity of trifluorothymidine (TFT) singly and in combination with other antiviral agents against human cytomegalovirus (HCMV) was evaluated by using an infectious center plaque reduction assay. The 50% inhibitory dose of TFT against six different patient HCMV strains was 0.57 (+/- 0.24, standard deviation) microM and ranged from 0.32 to 0.97 microM. The 50% inhibitory dose for the laboratory-adapted HCMV strain, AD-169, was 2.1 microM. When TFT (0.17 microM) was combined with human fibroblast interferon (25 U/ml), the combination was additive against all four HCMV isolates evaluated. Synergism was observed when TFT (0.17 microM) was combined with phosphonoformic acid (25 microM) for all strains studied or with acyclovir (20 microM) for three of the four clinical HCMV strains tested. Each of the three antiviral agents, when combined with TFT, exhibited additive effects against strain AD-169. TFT at concentrations of 0.5, 1.7, and 3.5 microM had an increasing inhibitory effect on uninfected human embryonic lung fibroblast (HEL) cell growth over 72 h, with 16% growth inhibition at 3.5 microM after 3 days. There was no increased toxicity to growing HEL cells when the paired antiviral agent combinations were evaluated. These findings suggest that TFT may be useful singly or in combination with other antiviral agents in treating HCMV infections.     

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.