乙型肝炎病毒感染HepG2细胞模型的构建

目的:构建乙型肝炎病毒(HBV)感染的细胞模型.方法:将携带1.2拷贝HBV基因的重组腺病毒感染HEK293细胞进行包装、扩增并分离纯化.将扩增得到的HBV感染HepG2细胞,用ELISA法检测感染后第4天培养上清中的HBsAg,Real-time RT-PCR检测HBV mRNA.结果:ELISA检测结果显示,HepG2细胞感染HBV后上清液中HBsAg呈阳性.Real-time RT-PCR可以检测到HBV mRNA.结论:HBV能在HepG2细胞中表达复制和表达.HBV感染的HepG2细胞模型构建成功.     

乙肝患者血清中丁肝病毒标志物检测的临床意义

目的 探讨乙型肝炎病毒(HBV)与丁型肝炎病毒(HDV)重叠感染后血清病毒性肝炎标志物的变化,为临床对乙肝患者病情的观察与治疗提供依据.方法 对156例HBsAg携带者,用ELISA法检测HBV和HDV免疫血清标志物(HBVM:HBsAg、抗-HBs、HBeAg、抗-HBe和抗-HBc;HDVM:HDAg和抗-HD),用PCR-荧光探针法检测HBV-DNA指标.结果 156例HBsAg携带者血清HDVM检出率为10.26%,HBV复制活跃组HDVM检出率为3.13%,较HBV复制缓慢组HDVM检出率21.67%明显低(P<0.001),HBV重叠感染HDV患者血清中HBV-DNA的检出率均明显低于单一HBV感染者,P<0.05.结论 HBV重叠感染HDV后,HDV复制可抑制HBV的复制和表达.     

白细胞介素-35对乙型肝炎病毒复制的影响研究

目的探讨白细胞介素(IL)-35对乙型肝炎病毒(HBV)复制的影响。方法将HBV感染性克隆pHBV1.3及其启动子pHBV-Luc分别转染HepG2细胞,加入不同浓度的重组人IL-35蛋白,采用酶联免疫吸附试验(ELISA)检测细胞上清液中乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)水平;荧光定量聚合酶链反应检测细胞闭合环状DNA(cccDNA)的水平;采用Luminometer荧光检测仪分析HBV启动子活性的变化。结果 IL-35能够抑制细胞上清液中HBsAg和HBeAg的水平及细胞内HBV cccDNA的拷贝数,并下调HBV启动子的活性。结论 IL-35能够在HepG2细胞中抑制HBV的复制。     

血清中HBsAg滴度与HBeAg消长关系的探讨

乙型肝炎系由乙肝病毒(HBV)所致的一种常见的、多发性传染病。而HBeAg又与多聚人血清白蛋白受体(PHSA-r)呈显著的平行关系。HBeAg阳性在乙肝患者中，由于针对HBeAg的淋巴细胞所介导的免疫应答导致的感染持续及肝细胞损伤叫。鉴于HBeAg与HBV复制和传染性及其所致肝细胞损伤的关系，我们对689例血清标本同步检测乙肝两对半和HBsAg滴度，凡HBsAg阳性血清用中和试验方法进行滴度定量，现将结果报告如下。     

N-乙酰半胱氨酸体外抗乙肝病毒作用

目的：探讨NAC体外抗乙肝病毒活性及其机制。方法：体外以转染了乙肝病毒的HepG2-2．2．15细胞株作为实验对象，培养细胞中加入不同浓度的NAC(0，3，10，30mmol／L)。用酶联免疫吸附法(ELISA)测定培养上清液中的乙肝表面抗原(HBsAg)、e抗原(HBeAg)。半定量PCR测定细胞内及培养上清液中的HBV DNA的变化，RT-PCR分析细胞内HBV表面抗原mRNA的变化。结果：NAC对HBsAg、HBeAg均有抑制作用，但NAC对HBsAg的抑制较HBeAg强，抑制率达90％左右。且抑制作用具有剂量和时间依赖性。半定量PCR结果显示细胞内的HBV DNA含量随着NAC浓度的增加而升高，而培养上清中的HBV DNA的含量降低。RT-PCR结果表明细胞内HBsAg mRNA没有显著变化。结论：NAC体外具有抗HBV活性，其抑制作用发生在转录后水平。     

慢性乙肝与携带者血清HBV DNA载量与血清HBeAg的相关性分析

目的 了解慢性乙肝与携带者血清HBV DNA病毒载量与血清HBeAg的相关性,为临床诊断和治疗提供依据.方法 采用酶联免疫实验和荧光定量聚合酶链反应(FQ-PCR)分别检测602例慢性乙型肝炎患者和携带者的血清标志物和HBV DNA载量水平,统计分析血清标志物水平与HBV DNA病毒载量水平的相关性.结果 HBeAg和HBV DNA之间有较好的一致性和相关性,HBsAg+ HBeAg+HBcAb+(大三阳)的HBV DNA阳性率及HBV DNA平均含量明显高于HBsAg+ HBeAb +HBcAb+(小三阳);且高于其他模式组合.HBeAg阳性慢性乙肝与携带者的HBV DNA载量升高例数均高于HBeAg阴性慢性乙肝与携带者,差异有统计学意义.结论 慢性乙肝与携带者血清中HBV DNA载量与HBeAg有很好的相关性,对诊断和治疗有重要的指导意义.     

HBV DNA载量与HBsAg、HBeAg和ALT水平的相关性

目的 了解HBV DNA载量HBsAg、HBeAg和ALT水平的相关性.方法 对148例患者采用荧光定量PCR法检测血清HBV DNA,用化学发光法检测血清HBsAg和HBeAg,用比色法检测血清ALT.结果 HBV DNA载量与HBeAg呈正相关(P<0.01),但与HBsAg和ALT无相关性.结论 定量检测HBV DNA可直接反映HBV在体内的复制情况,ALT水平的升高提示有肝脏病理性损伤.     

HBV血清标记物不同组合模式与荧光定量结果之间的关系

目的：对HBV血清标记物不同组合模式与HBVDNA定量结果进行对比分析。方法：采用ELISA方法检测乙肝病毒血清标记物,荧光定量PCR检测HBV—DNA,比较二者关系。结果：HBsAg、HBeAg、HBcAb阳性组、HB—sAg、HBeAg阳性组及HBsAg、HBsAb、HBeAg、HBcAb阳性组HBV—DNA检出率和平均水平含量均较高。结论：荧光定量PCR检测HBV—DNA能准确反映体内HBV真实感染和复制情况,同时进行HBV血清标记物的检测与HBV—DNA荧光定量PCR的检测,更有利于HBV临床感染上的诊断、对治疗方案的选择及疗效评价。     

慢性乙型肝炎抗病毒治疗进展（VCD）

根据2006年全国人群乙型肝炎血清流行病学调查结果显示，我国人群1—59岁人群中HBsAg携带率已从1992年的9．75％降至7．18％，按目前HBsAg携带率推算，我国仍然有HBsAg携带者约9300万人。HBV感染临床可分为不同类型，按照我国发布的《中国慢性乙型肝炎防治指南》分为慢性HBV携带者、慢性乙型肝炎（又分为HBeAg阳性和阴性慢性乙型肝炎）、非活动性HBsAg携带者、隐匿性慢性乙型肝炎、肝炎肝硬化及乙型肝炎相关肝细胞肝癌（HCC）。     

隐匿性乙型肝炎的临床意义

乙型肝炎病毒(HBV)是一种嗜肝细胞病毒，多数感染者可无临床症状，血清标志物(HBV—M)检测是乙型肝炎感染的重要依据。而HBsAg的阴性及抗HBs的出现一直认为HBV清除和临床痊愈的标志。随着分子生物学等技术在病毒检测中的应用，近年来发现部分复制水平较低的乙型肝炎病毒携带者血清中检测不到HBV表面抗原(HB-sAg)，这种感染状态可发生于抗HBs或抗HBc阴性的患者；     

Hepatitis B virus DNA and hepatitis B surface antigen levels in chronic hepatitis B

Despite universal vaccination, chronic hepatitis B (CHB) continues to be a major health burden worldwide, with an estimated 350–400 million people infected with the virus. Over the past decade, rapid progress has been made with regards to antiviral therapy for CHB, from conventional interferon to pegylated interferon, and with the earliest oral agent lamivudine to the current, more potent drugs such as entecavir and tenofovir. There have also been new developments in the diagnostic and monitoring tools for CHB. Qualitative hepatitis B surface antigen (HBsAg) testing has been used to diagnose patients infected with CHB. More recently, quantitative HBsAg titers have been used to predict treatment outcome when measured at baseline or early into treatment. The progress on the use of hepatitis B virus (HBV) DNA levels has been more rapid. Serum HBV DNA levels have been shown to be important in the natural history of CHB infection, with higher levels being significantly associated with the development of cirrhosis and hepatocellular carcinoma. For patients receiving antiviral therapy, the baseline and early on-treatment HBV DNA levels are important in determining treatment outcomes. Monitoring of HBV DNA levels during therapy will allow for early detection of drug resistance. The end-of-treatment and post-treatment HBV DNA levels have been demonstrated to be important indicators of treatment success and relapse, respectively. With newer and more powerful antiviral agents, and with the development of quantitative assays that are highly sensitive, further studies are needed to optimize the use of these tools and agents in the modern management of CHB.     

慢性HBV感染者肝组织HBV cccDNA的定量检测

目的建立一种肝活检组织中HBV共价闭合环状DNA（cccDNA）的定量检测方法。方法待检肝组织标本共21份，来源于江苏省人民医院肝脏手术患者，包括19份慢性HBV感染，其中HBeAg（＋）标本10份，HBeAg（-）标本9份，4份非HBV感染为阴性对照组，取HBVDNA阳性的患者外周血作为rcDNA组。检测方法的主要步骤为肝组织经蛋白酶K和细胞裂解缓冲液消化后，用液相抽提法提取核酸，将提取的核酸溶液分为2等份。1份用特异性降解单链DNA的核酸酶加以消化，纯化后使用跨缺口引物和SYBR GreenⅠ荧光染料进行实时荧光定量PCR分析；另1等份则用以定量检测肝细胞看家基因（β-Globin）作为样本细胞参数。检测结果的特异性主要通过PCR反应产物的序列分析及rcDNA组结果的对照进行证实，HBeAg（＋）组和HBeAg（-）组cccDNA水平的差异通过两样本t检验进行分析。结果PCR产物经琼脂糖凝胶电泳分析显示扩增产物的碱基数为350bp左右，DNA测序分析提示产物与目的片段的序列符合率为99％以上，且以rcDNA为对照的结果均为阴性，排除最有可能造成假阳性的rcDNA对结果的干扰。本方法对10mg HBeAg（＋）肝组织标本的cccDNA的检测阳性率为100％。血清HBeAg（＋）的肝组织样本的平均HBV cccDNA水平高于HBeAb（＋）的肝组织标本（P〈0．05）。结论通过上述三种途径证实了本文所建立方法的特异性。应用SYBR GreenⅠ荧光染料和β-Globin作为样本细胞参数所建立的实时荧光定量PCR方法检测肝细胞内HBV cccDNA，具有较高的特异性、敏感性，且成本较低的特点。     

Hepatitis B virus,hepatitis C virus

Medical workers should have anti-HBV antibody to protect HBV infection in the hospital. If they have not anti-HBV antibody, they should receive HBV vaccination. HBV vaccination program is as follows: 10 micrograms, s.c., 0, 1, 6 months. In case of HBV contamination, 1,000 IU hepatitis B immune globulin(HBIG) and/or 10 micrograms HB vaccine should be administered judging from HBV markers of contaminated subjects and HBV load of patients. The HCV vaccine is not available. In case of HCV contamination, it is unnecessary to treat just after accident. If acute hepatitis C is evolved in those subjects during follow-up, it is recommended to treat with interferon. Eradication of HCV by interferon among patients with acute hepatitis C will be almost 100%.     

Management of hepatitis B virus

Hepatitis B is a common problem worldwide with serious sequelae. Despite the explosion of new agents, management has grown even more complicated. The treatment paradigm is evolving from limited therapy to lifelong viral suppression in several populations. This shift has been a direct result of not only well-tolerated oral medications, but also the increasing recognition that active viral replication leads to untoward events such as cirrhosis, liver failure and hepatocellular carcinoma. However, therapy is not without risk, which includes side effects, cost and drug resistance. Controversy surrounds several clinical questions, including which patients are eligible for therapy, which treatment is optimal and at what point may therapy be discontinued. This commentary will discuss these questions as well as the limitations of the literature used to support our current treatment recommendations.     

Classifying hepatitis B virus genotypes

In 1988, hepatitis B virus (HBV) was classified into four genotypes by a sequence divergence in the entire genome exceeding 8%, and designated by capital letters of the alphabet from A to D. There are seven genotypes of HBV (A-G) at present, and an eighth is on the horizon. They have an uneven geographical distribution, and only a few of them are prevalent in a given area of the world. Thus genotype A is frequent in northwest Europe, Sub-Saharan Africa, India and the North, Central and South America, B as well as C are common in Southeast Asia and Oceania, and D is prevalent in the Mediterranean area, Central Asia and South America. Genotype E is restricted to West Africa, and F is localized in Central and South America. The distribution of genotype G added to the alphabet list very recently has yet to be determined. Coinfection with HBV of distinct genotypes is not infrequent and found in about 10% of infected individuals, and is responsible for intertypic recombination of HBV genomes. The mutation for a stop codon in the precore region (G1896A) for aborting the translation of hepatitis B e antigen (HBeAg) is prohibited in HBV genomes of genotype A, as well as some of genotypes C and F, because they possess C at position 1858 that makes a Watson-Crick pair with G at position 1896. Hence, seroconversion to antibody to HBeAg is forbidden or delayed in individuals who carry them. Evidence is accumulating as regards the influence of HBV genotypes on the progression of chronic hepatitis B and response to antiviral therapies. HBV isolates even of the same genotype can differ in virological and clinical characteristics, and therefore, the genotype needs to be classified further into subtypes, especially if they are clinically relevant.     

各类慢性乙型肝炎患者HBV前S2抗原与HBV感染血清标志物及HBVDNA的关系

目的探讨乙型肝炎病毒(HBV)前S2抗原(preS2Ag)与HBV感染5项标志物(HBV-M)、HBV DNA之间的关系及对慢性乙型肝炎分类诊断的临床意义。方法对584例各类慢性乙型肝炎患者血清采用放射免疫法(R IA)检测preS2Ag,用酶联免疫吸附试验(ELISA)检测HBV-M(HBsAg、抗HBs、HBeAg、抗HBe、抗HBc),用聚合酶链反应(PCR)检测HBV DNA。结果慢性乙型肝炎轻度、中度、重度及肝炎后肝硬化患者preS2Ag的检出率分别为45.98%、67.10%、83.87%及92.86%;preS2Ag在HBeAg阳性、HBV DNA>105拷贝/mL患者中检出率明显高于HBeAg阴性、HBV DNA<105拷贝/mL患者,差异有统计学意义(P<0.001)。结论preS2Ag的检出意味着病毒有复制或有传染性;开展preS2Ag的检测有助于慢性乙型肝炎的分类、慢性乙型肝炎急性发作和预后的判断。     

各类慢性乙型肝炎患者HBV前S2抗原与HBV感染血清标志物及HBV DNA的关系

目的探讨乙型肝炎病毒（HBV）前S2抗原（preS2Ag）与HBV感染5项标志物（HBV-M）、HBV DNA之间的关系及对慢性乙型肝炎分类诊断的临床意义。方法对584例各类慢性乙型肝炎患者血清采用放射免疫法（RIA）检测preS2Ag,用酶联免疫吸附试验（ELISA）检测HBV-M（HBsAg、抗HBs、HBeAg、抗HBe、抗HBc）,用聚合酶链反应（PCR）检测HBV DNA。结果慢性乙型肝炎轻度、中度、重度及肝炎后肝硬化患者preS2Ag的检出率分别为45.98%、67.10%、83.87%及92.86%;preS2Ag在HBeAg阳性、HBV DNA〉105拷贝/mL患者中检出率明显高于HBeAg阴性、HBV DNA〈105拷贝/mL患者,差异有统计学意义（P〈0.001）。结论preS2Ag的检出意味着病毒有复制或有传染性;开展preS2Ag的检测有助于慢性乙型肝炎的分类、慢性乙型肝炎急性发作和预后的判断。     

Precore wild-type hepatitis B virus with G1896 in the resolution of persistent hepatitis B virus infection

OBJECTIVE: Factors influencing the resolution of persistent hepatitis B virus (HBV) infection were sought for. METHODS: The loss of hepatitis B surface antigen (HBsAg) from serum was correlated with mutations in HBV DNA for a hepatitis B e antigen (HBeAg)-minus phenotype in patients infected with HBV genotype C and positive for HBeAg at presentation. RESULTS: HBeAg turned negative in all the 22 patients in whom HBV infection resolved, but only in 11 of the 25 patients with severe liver diseases (100 vs. 44%, p = 0.0001). The precore wild type (G1896) persisted significantly more frequently in the 22 patients in whom HBV infection resolved than in the 11 patients who developed decompensated liver cirrhosis or hepatocellular carcinoma (15/22 or 68% vs. 1/11 or 9%, p = 0.005). The double mutation in the core promoter (T1762/A1764) was comparably frequent in the two groups of patients at presentation (14/22 or 64% vs. 7/11 or 64%) and >15 years thereafter (18/22 or 82% vs. 10/11 or 91%). CONCLUSION: The precore wild type (G1896) would seem to facilitate the resolution of HBV infection, while the precore mutant (A1896) may induce severe liver diseases in patients with HBeAg-positive chronic hepatitis who have lost HBeAg from serum.     

Quantitative analysis of hepatitis B virus precore mutant in hepatitis type B.

Active liver disease has been detected in chronic hepatitis B after seroconversion from positive HBe antigen to positive anti-HBe antibody. Active replication of HB virus (HBV) containing a precore stop-codon mutation has been implicated in this condition. The usual methods, such as direct sequencing, to characterize the responsible mutant of HBV are not suitable for routine clinical use. Here we employed the competitive mutation site specific assay (CMSSA) to detect precore mutant HBV-DNA in patients with positive HB surface antigen. In patients with HBe antigen, precore mutant HBV-DNA was significantly higher than in patients with HBe antibody. The level of precore mutant HBV-DNA in patients with elevated serum ALT was significantly higher than in patients with normal serum ALT. Sex, age and the level of serum HBV-associated DNA polymerase levels were not correlated with levels of precore mutant HBV-DNA. Ten of 11 negative patients for the precore mutant by polymerase chain reaction followed by restriction fragment length polymorphism assay (PCRRFLP) were positive for the precore mutant by CMSSA. These results suggest that the precore mutant has already emerged in the HBeAg-positive phase as determined by CMSSA, which is more sensitive than PCR-RFLP and is useful for evaluating the clinical course of patients with chronic hepatitis B.