HCV不同基因型对慢性丙型肝炎抗病毒治疗疗效的影响

目的探讨干扰素联合利巴韦林治疗慢性丙型肝炎患者临床疗效的相关影响因素.方法对110例慢性丙型肝炎患者进行PEG-IFNα-2a 180μg/周联合RBV 800~1200 mg/d的标准治疗,并完成治疗后24周随访,同时分析HCV基因型、HCV-RNA定量对持续病毒学应答(SVR)的影响.应用罗氏试剂检测丙肝病毒定量,应用反转录-套式聚合酶链反应(PT-PCR)法检测HCV基因型.结果在110例进行标准联合治疗、并完成治疗后24周随访的慢性丙型肝炎患者中,68例患者获得SVR.其中,HCV基因1b型感染者SVR率为51.4%(37/72),HCV基因N-1b型感染者SVR率为81.5%(31/38);基线HCV-RNA水平1.0×10~5IU/m L的患者SVR率为71.2%(42/59),基线HCV-RNA水平≥1.0×10~5IU/m L的患者SVR率为50.9%(26/51).结论非1b基因型SVR率高于1b基因型;基线低病毒RNA水平(血清HCV-RNA水平1.0×10~5IU/m L)SVR率高于基线高病毒RNA水平(血清HCV-RNA水平≥1.0×10~5IU/m L).     

乳腺癌血清中MMP-7自身抗体的检测

目的运用ELISA技术测定乳腺癌血清中MMP-7自身抗体,从而推测血清中MMP-7自身抗体对乳腺癌的诊断价值。方法建立最佳优化条件,用间接ELISA检测39例正常人血清、43例乳腺癌患者血清中MMP-7自身抗体的存在情况,进而对检测方法的诊断价值进行评估。结果乳腺癌患者血清MMP-7自身抗体水平明显高于正常人,P〈0.01;ROC曲线分析表明,当选OD450值=1.344为cutoff值时,MMP-7自身抗体用于诊断乳腺癌的敏感性为76.7%,特异性为82.1%。     

乙型肝炎及慢性丙型肝炎患者混合感染庚型肝炎病毒的致病性研究

为探讨庚型肝炎病毒(GBV-C/HGV)感染有无致病性,利用GBV-C/HGV不同基因区序列设计并优选出5套引物,建立起灵敏、特异的HGV逆转录套式聚合酶链反应(RT-nPCR)检测方法.应用此方法对临床上诊断为乙型肝炎及慢性丙型肝炎的患者,按照其血清中HGV RNA的阳性与否分组,并进行病例对照研究.结果显示,单独HBV或HCV感染的肝炎患者与混合HGV感染的肝炎患者的血清肝功能异常指标无显著性差异(P>0.05).因此,病毒性肝炎患者混合感染HGV后,未发现加重HBV或HCV感染的肝功能异常程度,不支持HGV有明显的嗜肝致病性.     

反向线性探针杂交技术检测慢性乙型肝炎病毒拉米夫定耐药研究

目的:建立起简单、快速、灵敏、准确的慢性乙型肝炎病毒拉米夫定耐药位点的反向线性探针(reverse line probe,RLP)检测方法.方法:根据HBV野生及耐药基因序列设计通用探针、3'、5'对称加有poly-C的特异性探针和5'标记生物素的扩增引物.将探针线性固定在硝酸纤维膜上,使HBV PCR扩增产物与探针进行杂交.通过优化杂交条件,建立RLP检测方法.利用该方法对重庆地区86个慢性乙肝病人进行检测,同时与直接测序结果比较.结果:半巢式PCR可对103拷贝/ml的血清样本进行有效特异扩增,新建的RLP检测方法可对PCR扩增产物在1 ng/ml以上,或血清样本中突变型DNA占野生型DNA比例为5％以上的均可有效检测,86例临床的检测灵敏度为100％,野生型和耐药型的检测准确性分别为98.09％(103/105)、100％ (43/43),与直接测序法比,RLP检测野生与耐药混合型准确性更好.结论:反向线性探针杂交检测方法检测HBV拉米夫定耐药位点方便、灵敏、准确,是HBV拉米夫定治疗有效的监控工具,该方法适合临床应用.     

用非同位素标记的pAV 401探针进行分子杂交鉴定马铃薯纺锤块茎类病毒

用Eoehringer Mannheim Biochemica的pAV401探针,以分子杂交的方法鉴定了马铃薯和番茄中的马铃薯纺锤块茎类病毒(PSTV)。试验表明当用探针浓度为200ng/ml, β-半乳糖苷酶为100 mU/ml,即酶结合物稀释至1:100时,可以测定出提纯PSTV—RNA最低浓度为390pg/斑点,感染PSTV的马铃薯和番茄叶片汁液最高稀释浓度为1:128。样品提取方法试验表明,以酚法教果为最佳。健康马铃薯汁液对提纯PSTV—RNA鉴定效果无明显影响。样品汁液在稀释前予先变性对提高杂交效果无明显作用。本文对Boehringer pAV401探针检测PSTV的灵敏度,所用探针浓度以及显色系统进行了讨论和评价。     

核酸分子杂交检测黄瓜花叶病毒

为检测黄瓜花叶病毒的存在,应用Northernblot,将提取的黄瓜花叶病毒RNA样品,经甲醛变性凝胶电泳分离,使RNA解离成单链,然后用上行毛细管转移法,将凝胶上的单链RNA片段转移到尼龙膜上.经适当洗涤、干燥后,在烤箱中80℃保温2h,用于杂交.以带有黄瓜花叶病毒目的片段的细菌质粒制备DNA探针,用放射性同位素32P标记探针DNA,100℃变性处理用于杂交的探针,然后进行杂交.结果表明,在杂交膜上显示与特异性探针结合的阳性RNA条带,检测到了黄瓜花叶病毒的存在.     

62例肝功能异常学龄儿童输血传播病毒的检测结果

目的：分析血丙氨酸氨基转移酶(ALT)升高的学龄儿童(7—12岁)输血传播病毒(TTV)感染情况，并对其致病性及与其它肝炎病毒感染的关系做初步探讨。方法：应用巢式聚合酶链反应技术(Nested PCR)，对62例体检发现ALT升高和75例ALT正常的学龄儿童血标本进行TTV DNA扩增，通过凝胶电泳分离扩增产物；用ELISA方法同时检测甲、乙和丙型肝炎病毒感染的血清标志。结果：ALT升高中，TTV DNA检出率为12．9％(8／62)，其中3例HBsAg同时阳性(4．8％)，1例与抗—HCV同时阳性(1．6％)；ALT正常中，TTV DNA检出率为2．7％(2/75)，两差异具有显性意义(P＜0．05)。结论：肝功能异常学龄儿童存在较高的TTV感染率且有可能致肝功能损害。TTV与肝炎病毒可重叠感染同一儿童。     

地高辛掺入法检测HCV RNA含量在肝病诊断中的应用

目的：研究血清HCVRNA含量与HCV感染者肝病程度的关系。方法：应用地高辛掺入法检测血清HCVRNA含量。在PCR过程中将DIG－dUTP掺入到PCR产物中去，再用PCRELISA法检测PCR产物，结合标准参考样本而达定量目的。结果：丙肝后肝硬化组血清HCVRNA含量显著高于丙型肝炎组。结论：地高辛掺入法检测血清HCVRNA含量可以为HCV感染者的病情判断提供科学依据。     

丙型肝炎病毒及其检测的研究现状与进展

丙型肝炎分布于世界各地。我国是世界上丙型肝炎的高流行区之一。据初步调查,一般人群中丙型肝炎病毒的感染率为2—4％,受血人群和职业献血员的感染率更高。因此丙型肝炎病毒(Hepatitis C virus HCV)的研究,检测方法及试剂的研制已成为人们关心的重要课题。现就HCV的分子生物学特性及其检测方法研究的现状与进展作一综述。     

蓝舌病病毒内蒙古分离株VP2基因5′端序列分析及探针制备

从内蒙古巴盟地区分离的蓝舌病病毒株(BTV—NM)提取总RNA，经反转录PCR扩增VP2基因5′端片段，并构建至pGEM—T载体中。序列测定后，将这一序列与蓝舌病毒澳大利亚株VP2基因5′端进行比较分析：同源性为40％。通过PCR法标记克隆的cDNA片段，制备地高辛标记探针，与粗提的蓝舌病发病羊病毒RNA进行Northernblot杂交，并作敏感性试验，结果表明此探针对蓝舌病毒内蒙古分离株具有特异性，可检测出50pg的病毒RNA。     

HCV非结构基因NS5分子生物学研究进展

丙型肝炎病毒(HCV)非结构基因5(NSS)区是RNA依赖的RNA聚合酶(RDRP)及一种磷蛋白的编码区,与HCV的复制、致病及抗药性关系密切。就HCV NS5区有关的分子生物学研究进展进行了综述。     

丙型肝炎病毒感染与ras,p53基因表达的相关性

为了探讨丙型肝炎病毒（ＨＣＶ）感染与肝细胞癌（ＨＣＣ）的关系以及ＨＣＶ可能的致癌机理，采用免疫组织化学方法及巢式ＰＣＲ法检测了１３６例肝细胞癌等肝病组织中的ＨＣＶＮＳ３抗原、ＨＣＶＲＮＡ及Ｐ２１、Ｐ５３蛋白。结果表明，肝细胞癌及癌周肝组织中有ＨＣＶＮＳ３抗原及ＨＣＶＲＮＡ检出，支持ＨＣＶ与ＨＣＣ的关联。Ｐ２１在ＨＣＣ、肝炎后肝硬化、慢性肝炎、体质性黄疸各组中的检出率随病变的加重而逐渐增高，在ＨＣＣ的癌及癌周组织中Ｐ２１呈致密的过量表达，提示ｒａｓ癌基因的激活在ＨＣＣ的发生过程中起一定作用。Ｐ５３的阳性率较Ｐ２１低，但ｐ５３的突变似乎也是肝癌发生的协同因素之一。组织中Ｐ２１的过量表达与ＨＣＶＮＳ３抗原阳性检出呈正相关，ＨＣＶＮＳ３抗原与Ｐ２１的这种关联提示，ＨＣＶ感染作为ＨＣＣ的密切相关因素之一，可能通过激活某些癌基因或使某些抑癌基因突变而致肝细胞癌变     

RNA interference in adult mice

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.     

Human aminopeptidase N is a receptor for human coronavirus 229E.

Human coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections. Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.     

HCV感染与多种自身抗体高检出率的关系

为探讨丙型肝炎病毒（ＨＣＶ）感染后体内出现的自身抗体的病理意义及其引起的自身免疫的发生机制。方法：对１６１例ＨＣＶ感染者进行了八种自身抗体（抗核抗体、类风湿因子、抗甲状腺球蛋白抗体、抗甲状腺微粒体抗体、抗双链ＤＮＡ抗体、抗ＲＮＰ抗体、抗Ｓｍ抗体、抗精子抗体）的检测。结果：有５２例检出６９项次自身抗体,自身抗体检出率为３２．３％,显著高于健康人对照组（Ｐ＜０．００５）（未计抗精子抗体）。结论：ＨＣＶ感染可能是诱发自身免疫反应的一个重要因素     

Interferon modulation of cellular microRNAs as an antiviral mechanism

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.     

Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.     

Modelling how ribavirin improves interferon response rates in hepatitis C virus infection

Nearly 200 million individuals worldwide are currently infected with hepatitis C virus (HCV). Combination therapy with pegylated interferon and ribavirin, the latest treatment for HCV infection, elicits long-term responses in only about 50% of patients treated. No effective alternative treatments exist for non-responders. Consequently, significant efforts are continuing to maximize response to combination therapy. However, rational therapy optimization is precluded by the poor understanding of the mechanism(s) of ribavirin action against HCV. Ribavirin alone induces either a transient early decline or no decrease in HCV viral load, but in combination with interferon it significantly improves long-term response rates. Here we present a model of HCV dynamics in which, on the basis of growing evidence, we assume that ribavirin decreases HCV infectivity in an infected individual in a dose-dependent manner. The model quantitatively predicts long-term response rates to interferon monotherapy and combination therapy, fits observed patterns of HCV RNA decline in patients undergoing therapy, reconciles conflicting observations of the influence of ribavirin on HCV RNA decline, provides key insights into the mechanism of ribavirin action against HCV, and establishes a framework for rational therapy optimization.