Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) ...     

乙型肝炎病毒共价闭合环状DNA的研究进展

共价闭合环状DNA(covalently dosed circular DNA,cccDNA)在HBV的复制及感染状态的建立过程中起着非常重要的作用,现将cccDNA的研究进展总结如下. 一、HBV cccDNA的检测方法 1.PCR法:PCR法包括普通PCR和定量PCR.由于松弛环状DNA(relaxed circular DNA,rcDNA)在负链和正链上存在缺口,我们可以设计一对跨越两个缺口的引物,由于cccDNA有完整的双链,可以被有选择地扩增.但起始模板量不能太高,否则rcDNA也有可能被扩增,Kock等[1]发现,每个PCR反应管中HBV DNA量在6×105拷贝/ml时能达到最佳的灵敏度与选择性.     

乙型肝炎病毒感染的肝癌患者肝组织中乙型肝炎病毒cccDNA的检测

HBV属嗜肝DNA病毒家族,其基因组是双链松弛环状DNA(rcDNA),相对分子量为(1.6～2.0)×109,全长约为3.2 kb。在HBV的生命周期中,共价闭合环状DNA (cccDNA)是HBV的原始复制模板,对HBV的复制及感染状态的建立具有十分重要的意义。清除HBV cccDNA是目前抗HBV药物研究的一个重要目标。我们对天津市第三中心医院收治的20例HBV感染的肝癌患者肝组织(包括癌     

54例慢性乙型肝炎患者肝组织乙型肝炎病毒共价闭合环状DNA与血清HBsAg定量检测结果分析

目的 检测慢性乙型肝炎患者肝组织HBV共价闭合环状DNA (cccDNA)和血清HBsAg,分析两种定量指标之间及其与血清HBV DNA载量的相关性.方法 应用PSAD消化+滚环扩增+跨缺口实时荧光PCR方法,定量检测54例慢性乙型肝炎患者甲醛固定石蜡包埋肝组织HBV cccDNA水平;用化学发光试剂定量检测患者血清HBsAg.用Pearson检验及直线回归分析方法 对数据进行分析.结果 患者肝组织HBV cccDNA与血清HBsAg定量水平之间呈正相关(r=0.459,P＜0.01),但与血清HBV DNA载量相关性无统计学意义;血清HBsAg定量水平与血清HBV DNA载量呈正相关(r=0.328,P＜ 0.05),与病毒复制效率呈负相关(r=-0.373,P＜0.05).结论 慢性乙型肝炎患者肝组织HBV cccDNA载量与血清HBsAg定量水平相关,结合血清HBVDNA定量检测,可以更全面的反映HBV的复制水平,评价抗病毒疗效.     

Effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients

AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.
METHODS： Seventy-one patients received lamivudine （n = 35）, or sequential therapy with lamivudine- interferon alpha 2b （IFN-α 2b, n = 24） for 48 wk, or IFN-α 2b （n = 12） for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.
RESULTS： Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively （P 〈 0.05）. Seventeen out of the 71 patieots developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients （3.0 log vs 1.6 log, P = 0.0407）. Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound （4.6 log vs 5.4 log, P = 0.0472）. HBV genotype C accounted for 85.9% （n = 61）, and genotype B for 14.1% （n = 10）, respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B （2.1 log vs 1.9 log）.
CONCLUSION： Forty-eight week sequential lamivudine- INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.     

改良聚合酶链反应检测HBV共价闭合环状DNA

目的:建立一种基于聚合酶链反应(PCR)的简便快速、具有较高敏感性和特异性的检测乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)的方法.方法:分别提取HepG2.2.15细胞内的cccDNA及培养上清中的松驰环DNA(rcDNA)样品,试剂盒纯化;设计2对特异性引物,其扩增区域跨越rcDNA单链区;设计2对非特异性引物,扩增区域位于rcDNA双链区.经单链特异性绿豆芽核酸酶(MBN)分别消化cccDNA及rcDNA样品;以特异性引物和非特异性引物对消化前后的两种样品分别进行PCR扩增,并改变PCR扩增参数如底物数量、循环次数等,观察特异性引物能否顺利扩增消化后的cccDNA,同时又不扩增消化后的rcDNA.HBV基因组质粒样品作为对照.此外还采用实际乙型肝炎患者体内病毒样本检验此策略的实用性.结果:分别以非特异性引物和特异性引物扩增不同模板数的HBVrcDNA样品,2对非特异性引物可扩增出模板数在102以上的HBVrcDNA样品,2对cccDNA特异性引物也可以扩增出模板数在104以上的样品.特异性引物在PCR反应模板数较多时将不能区分消化前的rcDNA和cccDNA.不同数量HBVcccDNA和rcDNA模板在MBN消化前后,分别应用非特异性引物和特异性引物进行PCR扩增,发现不同数量的cccDNA模板分子经过MBN消化后,仍可用特异性引物和非特异性引物扩增出相应条带;rcDNA样品经过MBN消化后,非特异性引物可扩增出产物条带,而特异性引物无法扩增出条带.采用此种策略,我们发现慢性乙肝患者血清HBV核酸样品主要成份为rcDNA,并带有少量cccDNA,而肝细胞内HBV核酸样品富含cccDNA,与实际情况一致.结论:联合应用MBN选择性消化和cccDNA特异性引物的PCR检测法简便快速,敏感性和特异性均较满意.     

Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways

Hepatitis B virus (HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon (IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors (TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.     

RNA干扰抑制乙型肝炎病毒复制的实验研究

目的 以乙型肝炎病毒(HBV)核心区为靶位，构建表达小干扰RNA(siRNA)的质粒载体pSilencer3．1-Hlhygro，体外观察siRNA抗HBV的效果。方法 以HepG2 2．2．15细胞为靶细胞，利用脂质体Metafectene与表达siRNA的质粒载体pSilencer3．1-Hlhygro共转染，用定量聚合酶链反应检测细胞上清液中DNA，用逆转录聚合酶链反应检测HBV C-mRNA。结果 成功构建了表达siRNA的转录质粒载体，两条siRNA均可抑制HBV的复制，而且与siRNA浓度成正相关。结论 靶向HBV核心区的siRNA能抑制HBV的复制。     

乙型肝炎病毒共价闭合环状DNA的扩增与清除动力学

HBV基因组是一种松弛环状的双链DNA(relaxed circular DNA,rcDNA)分子,其两条链均不闭合,在病毒的复制过程中,病毒DNA进入宿主细胞核,两条链的缺口被补齐,形成超螺旋的共价闭合环状DNA(covalently dosed circular DNA,cccDNA).cccDNA是HBV在肝内进行复制的原始模板,虽然其含量较少,但对病毒在宿主细胞内感染状态的建立以及维持病毒在细胞内的复制过程具有十分重要的意义.     

商陆抗病毒蛋白不同结构域的抗乙型肝炎病毒活性

目的 比较全长和不同片段缺失型商陆抗病毒蛋白(PAP)基因真核表达质粒体外抗HBV作用及其细胞毒性作用.方法 将全长和不同片段缺失型PAP基因真核表达质粒用脂质体转染HepG2.2.15细胞,收获生长良好的HepG2.2.15细胞,转染前1 d,接种于24孔培养细胞板,培养20 h后,待细胞密度达到40%～50%时进行转染.细胞随机分为4组:pXF3H组,转染空质粒pXF3H作为对照;pXF3H-PAP_(12)组,转染全长PAP的真核表达质粒pXF3H-PAP_(12);pXF3H-PAP_(14)组,转染C端缺失25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(14);pXF3H-PAP3_(34)组,转染既缺失N端69个氨基酸又缺失C端25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(34).转染的质粒剂量为每孔1.0μg,终浓度为2.0μg/ml,质粒DNA(μg)和脂质体(μl)的比例为1:2.5,转染72 h后收集细胞及培养上清液.酶联免疫吸附法检测培养上清液HBsAg和HBeAg,荧光定量PCR检测HBV DNA水平,四甲基偶氮唑盐比色法检测各质粒对转染细胞的毒性作用.应用SPSS12.0软件包处理数据,两样本均数的比较采用t检验,率的比较采用χ~2检验.结果 对HBsAg、HBeAg、HBVDNA的抑制率,pXF3H-PAP_(14)组分别为56.3%、75.8%和61.7%,pXF3H-PAP_(12)组分别为61.4%、84.2%和63.2%,两组间差异无统计学意义.但pXF3H-PAP_(14)组细胞毒性 (抑制率为10.2%)明显低于pXF3H-PAP_(12)组(抑制率为27.1%),χ~2=7.7,P<0.01.pXF3H-PAP_(34)组无细胞毒性,但其抗HBV作用也丧失,对HBsAg、HBeAg、HBV DNA的抑制率分别为7.8%、11.0%、20.5%.结论 PAP的C端25个氨基酸与细胞毒性相关,与抗HBV活性无关;PAP的N端69个氨基酸与抗HBV活性相关.     

胡黄连总苷抑制乙型肝炎病毒复制作用的体外研究

目的 观察胡黄连总苷抑制HepG 2.2.15细胞内HBV共价闭合环状DNA(HBVeeeDNA)的作用效果及特点.方法 分别用50 mg/L胡黄连总苷或5 mg/L阿德福韦酯的细胞培养液作用于HepG 2.2.15细胞,加药后2 d、5 d收集细胞及培养上清液,实时荧光定量PCR方法检测上清液和细胞内HBV DNA、细胞内HBV cccDNA和前基因组RNA(pgRNA)水平,分别计算抑制率.均值比较用t检验.结果 胡黄连总苷作用2 d和5 d后,上清液HBV DNA抑制率为49.74%(t=4.723,P<0.05)和79.48%(t=7.512,P<0.05);细胞内HBV cccDNA抑制率为43.55%(t=5.216,P<0.05)和56.43%(t=7.262,P<0.05),总DNA抑制率为43.39%(t=4.137,P<0.05)和63.86%(t=7.861,P<0.05),pgRNA抑制率为54.72%(t=4.532,P<0.05)和56.08%(t=4.833,P<0.05).阿德福韦酯作用2 d和5 d后,上清液HBV DNA抑制率为25.56%(t=2.874,P<0.05)和92.44%(t=10.276,P<0.05);细胞内HBV cccDNA抑制率为18.54%(t=2.736,P<0.05)和47.19%(t=6.852,P<0.05),总DNA抑制率为21.20%(t=3.206,P<0.05)和71.47%(t=8.332,P<0.05),pgRNA抑制率为11.14%(t=1.761,P>0.05)和37.61%(t=3.632,P<0.05).结论 胡黄连总苷能明显抑制HepG 2.2.15细胞内HBV复制,尤其对cccDNA具有抑制作用,在作用时相上早于阿德福韦酯,可能存在不同的作用机制.     

Application of hepatitis B virus replication mouse model

AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model‘s value ...