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.     

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.     

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.     

Long-term clinical impact of occult hepatitis B virus infection in chronic hepatitis B patients

BACKGROUND/AIMS: Long-term clinical outcomes of occult hepatitis B virus (HBV) infection were studied. METHODS: Fifteen chronic hepatitis B patients were monitored for a median of 4.4 years (range 0.9-15.3) after hepatitis B surface antigen (HBsAg) seroclearance. Serum HBV DNA was measured by real-time detection polymerase chain reaction. Thirteen patients underwent liver biopsies at the end of follow-up and liver histology was evaluated by Ishak score. Liver HBV DNA was also measured for 12 patients. RESULTS: At the end of follow-up, HBV viremia was absent in 13 (87%) patients, and antibody titers to hepatitis B core antigen showed an inverse correlation with time from HBsAg seroclearance (r=-0.554; P=0.0040). However, all patients retained liver HBV DNA and tested positive for the covalently closed circular HBV DNA replicative intermediate. The hepatic HBV DNA loads had no relation to liver histology. Paired biopsies from 11 patients disclosed that each necroinflammatory score significantly improved after HBsAg seroclearance. Amelioration of liver fibrosis was also evident in eight (73%) patients (P=0.0391 by signed rank test). CONCLUSIONS: A long-standing but strongly suppressed HBV infection may confer histological amelioration after HBsAg seroclearance.     

核苷(酸)治疗下较短的cccDNA半衰期或给“乙肝治愈”点燃希望

乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)是HBV复制的模板,在缺乏直接靶向cccDNA治疗药物的情况下,阻断cccDNA的补充并尽快耗竭已有的cccDNA是实现慢性乙型肝炎彻底治愈的关键。既往研究认为cccDNA的半衰期很长,但近期的一项研究结果提示cccDNA池的更新周期仅为数月,远低于之前的预测。未来,随着新的、能够完全抑制HBV复制的抗病毒药物出现,cccDNA池将有望因其补充被完全阻断而被彻底清除,进而实现慢性乙型肝炎的病毒学治愈。     

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients

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Diagnostic strategy for occult hepatitis B virus infection

In 2008,the European Association for the study of the liver(EASL) defined occult hepatitis B virus infection (OBI) as thepresence of hepatitis B virus(HBV) DNA in the liver(with detectable or undetectable HBV DNA in the serum) of individuals testing hepatitis B surface antigen(HBsAg) negative by currently available assays.Several aspects of occult HBV infection are still poorly understood,including the definition itself and a standardized approach for laboratory-based detection,which is the purpose of this ...     

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) ...     

肝组织cccDNA水平与血清病毒学应答后治疗时间的关系

目的 探讨慢性乙型肝炎(CHB)患者肝组织cccDNA水平与外周血HBV DNA<1000 拷贝/ml后继续治疗时间的关系.方法 分别采用荧光定量PCR、酶联免疫吸附分析法检测58例CHB患者肝组织HBV cccDNA水平、肝组织和血清HBV DNA载量、HBV标志物,分析肝组织HBV cccDNA水平、肝组织总HBV DNA水平、HBeAg血清学转换与血清病毒学应答后继续治疗时间的关系.组间比较采用Nemenyi法,相关分析采用Spearman法.结果 肝组织HBVcccDNA水平在血清HBV DNA两阳性组间尢明显差异,而阴性组明显低于阳性组(χ2=9.6948,P<0.01;χ2=9.2824,P<0.01).35例达到血清病毒学应答后行肝活组织检查的患者,肝组织cccDNA水平随继续治疗时间的延长而降低(χ2≥6.4674,P<0.05),肝组织cccDNA水平在抗-Hbe(+)组明显低于HBeAg(+)组、HBeAg(-)/抗-Hbe(-)组(χ2=10.7482,P<0.01;χ2=11.7549,P<0.01).14例肝组织cccDNA水平低十检测限的患者,有12例已经发生HBeAg血清学转换,占抗-Hbe(+)组的2/3,其在血清病毒学应答后继续治疗时间平均为35个月,发生HBeAg血清学转换后继续治疗时间平均为30个月.结论 当患者发生血清病毒学应答后,肝组织cccDNA水平随继续治疗时间延长而降低;继续治疗35个月以上且血清抗-Hbe持续(+)30个月以上时,有2/3的患者肝组织cccDNA定量低于检测限水平.     

Measurement of hepatitis B virus core-related antigen as predicting factor for relapse after cessation of lamivudine therapy for chronic hepatitis B virus infection

Background

Prolonged lamivudine therapy has two major problems: breakthrough hepatitis during treatment and relapse of aminotransferase (ALT) after cessation of the therapy. The aim of this study was to examine factors that could predict ALT flare after stopping lamivudine therapy.

Methods

We analyzed 22 Japanese patients with chronic hepatitis B infection, in whom lamivudine therapy was stopped after HBV DNA level had been gone undetectable (<3.7 LGE/ml) during at least six consecutive months. The post-treatment followed up was carried for 28 months in median (range 9–41). HBV core-related antigen (HBcrAg) assay was assessed using newly developed assay.

Results

After cessation of lamivudine therapy, 11 patients (50%) had relapsed (reactivation of serum ALT >80 IU/l, relapsers) and remaining 11 (50%) did not relapse (non-relapsers). In the univariate comparison of relapsers versus non-relapsers, HBcrAg level at lamivudine cessation point (4.5 ± 1.0 versus 3.4 ± 0.9; p = 0.0145) has been shown as a significant predictive factor for non-relapse. All patients with HBcrAg <3.0 log U/ml at the cessation point had no ALT flares. Multivariate analysis on effects of 10 factors (age, sex, cirrhosis, pretreatment ALT level, HBV DNA level, HBcrAg level, mean months till undetectable HBV DNA, duration of undetectable HBV DNA and HBcrAg level at lamivudine cessation point), indicated that HBcrAg level at lamivudine cessation point <3.4 log U/ml was the only independent predictive factor for absence of the post-treatment relapse.

Conclusions

HBcrAg level at lamivudine cessation point might be useful as a prognostic predictor of response to lamivudine therapy cessation. The measurement of HBcrAg is a useful additional test for monitoring chronic HBV infection.     

An overview of occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection (OBI), alternatively defined as occult hepatitis B (OHB), is a challenging clinical entity. It is recognized by two main characteristics: absence of HBsAg, and low viral replication. The previous two decades have witnessed a remarkable progress in our understanding of OBI and its clinical implications. Appropriate diagnostic techniques must be adopted. Sensitive HBV DNA amplification assay is the gold standard assay for detection of OBI. Viral as well as host factors...     

Prevalence of occult hepatitis B virus infection

Occult hepatitis B virus(HBV) infection(OBI) is characterized by the persistence of HBV DNA in the liver tissue in individuals negative for the HBV surface antigen.The prevalence of OBI is quite variable depending on the level of endemic disease in different parts of the world,the different assays utilized in the studies,and the different populations studied.Many studies have been carried out on OBI prevalence in different areas of the world and categories of individuals.The studies show that OBI prevalence...     

MiR-122 in hepatitis B virus and hepatitis C virus dual infection

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.     

血清cccDNA与HBVDNA、YMDD变异及肝炎复发的临床关系

目的探讨cccDNA与病毒复制及拉米夫定耐药突变(YMDD)及肝脏病变的关系。方法采用分子信标PCR技术检测HBV携带者(ASC)、慢性乙型肝炎(CHB)、乙型肝炎肝硬化(LC)、肝癌(HCC)患者血清中cccD-NA与HBVDNA及YMDD突变。结果在283例HBV感染者血清中,cccDNA阳性123例(43.46%),均为HBVD-NA阳性标本;cccDNA与血清HBVDNA相关(x2=28.27,P<0.01)及ALT相关(x2=48.46,P<0.01)。60例接受拉米夫定治疗半年患者复查血清ALT、HBVDNA、YMDD及ccDNA,显示ALT异常32例(与cccDNA相关x2=48.46,P<0.01),HBVDNA阳性24例(与cccDNA相关x2=28.27,P<0.01),其中包括YMDD阳性18例与cccD-NA阳性16例(P=0.046)。结论血清cccDNA,是反映HBV复制及肝脏细胞损伤的血清标志。监测YMDD与血清cccDNA可以提示抗病毒治疗中HBV复制状态及病变进展情况。     

二十岁以下慢性HBV感染者HBVDNA与HBeAg的定量关系

目的:探讨20岁以下慢性乙型肝炎病毒(HBV)感染者血清中HBV DNA、乙型肝炎病毒e抗原(HBeAg)定量之间关系.方法:用实时荧光定量聚合酶链反应(FQ-PCR)及时间分辩荧光免疫分析(TRFIA)技术检测339例(1-20岁)慢性HBV感染者血清中HBVDNA、HBeAg含量,用速率法检测ALT水平.结果:HBeAg定量＞0.3 NCU/mL、HBV DNA定量＞105 copies/mL、而ALT水平正常者占总检测病例的92.3%;HBV DNA定量(对数值)与HBeAg定量之间存在正相关关系(r=0.769,P＜0.001)和线性回归关系(b=0.32,R2=0.59,P＜0.001).结论:20岁以下慢性HBV感染者血清中HBVDNA水平与HBeAg水平存在同时消长的关系,但是有极少患者例外.HBV DNA定量与HBeAg定量两种检测方法相结合应能够更客观地反映患者HBV感染状况,二者具有互补性.