HBIG,无环鸟苷,干扰素联合对慢性乙型肝炎抗病毒效应观察

本文报道血清ＨＢＶ复制标志阳性的慢乙肝５４例,随机分为治疗组及对照组各２７例进行ＨＢＩＧ、无环鸟苷、干扰素联合近、远期抗病毒效应观察。治疗组为无环鸟苷第一周按２５～２０ｍｇ／ｋｇ／ｄ计后改１７～１５ｍｇ／ｋｇ／ｄ×５３天,共６０天;人白细胞干扰素１×１０６Ｕ肌注每周３次×４周,后改１．０×１０６Ｕ肌注每周２次×６周,共１０周;ＨＢＩＧ４００Ｕ肌注隔日１次,共１０周,对照组仅给予一般“保肝”药物。其中治疗组１８例,对照组１９例进行治后半年到２年追踪观察,结果近、远期ＨＢｃＡｇ、ＤＮＡＰ、ＨＢＶ－ＤＮＡ阴转率治疗组均高于对照组,其中治疗组近、远期ＨＢｃＡｇ,ＨＢＶ－ＤＮＡ阴转率均达４０％以上,明显高于对照组（Ｐ＜０．０５～０．０１）,治疗组近、远期各有４例及２例ＨＢｓＡｇ阴转,而对照组则无一例阴转,从近、远期综合抗病毒效应观察,治疗组全阴率分别为３３．３％、４４．４％,而对照组分别为３．７９％及０％,Ｐ＜０．０１,治疗组无明显毒副反应。对比单用无环鸟苷,全阴率３１．８％;无环鸟苷加干扰素两药联合全阴率３７．５％,均有所提高,达到４４．４％,值得进一步研究。     

从乙型肝炎病毒表面抗原阳性母亲流产的胎儿查出乙型肝炎病毒标志

应用Southern blot杂交试验检测HBsAg及HBeAg均阳性母亲流产的9例胎儿肝细胞中HBV DNA的存在状态,并与其HBV血清学、免疫电镜及肝脏免疫组织化学的结果相比较。结果在3例胎肝高分子DNA中检出了整合的HBV DNA顺序,且此3例HBV DNA整合到胎肝细胞基因组并无特定部位,提示为随机整合。3例中2例的血清及肝匀浆都检出HBsAg颗粒,其胎肝细胞胞浆HBsAg也阳性;另1例受HBV感染的唯一标志是在胎肝细胞中存在着整合的HBVDNA。此外,另1例则仅胎肝细胞中HBsAg阳性而无整合的HBV DNA。在胎肝细胞中检出整合的HBV DNA进一步证实HBV子宫内传播途径的存在。     

免疫预防阻断乙型肝炎病毒母婴传播效果的观察

阻断乙型肝炎病毒(HBV)母婴传播是控制乙型肝炎的重大问题。为探讨免疫预防对阻断HBV母婴传播的效果及影响因素,对667例HBV表面抗原(HBsAg)阳性孕妇及其婴儿进行研究。这些孕妇按HBVe抗原(HBeAg)和HBVDNA检测结果,分为HBeAg阳性组及阴性组、DNA阳性组及阴性组;按是否于孕晚期注射乙型肝炎免疫球蛋白(HBIG),分为注射组及未注射组。婴儿于出生24h内均肌内注射HBIG100IU,并按0、1、6方案注射10μg重组酵母HBV疫苗;8～12月龄后随访婴儿,并进行HBV标志物(HBV-M)检测。667个婴儿中,20例感染HBV,免疫阻断失败率为3.0%。孕妇HBeAg阳性组免疫阻断失败率为8.7%,阴性组为0.2%,两组差异显著(P<0.001);两组婴儿对疫苗免疫应答率分别为83.0%和83.1%,无显著差异(P=0.988)。孕妇DNA阳性组免疫阻断失败率为8.1%,HBVDNA均≥6log10copies/ml。孕期注射与未注射HBIG组婴儿免疫阻断失败率分别为3.7%和2.7%,无显著差异(P=0.479);两组婴儿对疫苗免疫应答率分别为84.4%和82.4%,无显著差异(P=0.519)。孕妇HBeAg阳性注射HBIG组与未注射组的免疫阻断失败率分别为8.4%和8.9%,无显著差异(P=0.892)。孕妇HBeAg阴性注射与未注射HBIG组的免疫阻断失败率分别为0.0%和0.3%,也无显著差异(P=0.538)。11例免疫阻断失败的婴儿中,10例出生时血清HBsAg已为阳性;8～12个月后随访,HBsAg仍持续阳性,提示为宫内感染。本研究证实,孕期注射HBIG未能提高婴儿对HBV疫苗加HBIG的免疫阻断效果。宫内感染可能是疫苗加HBIG免疫阻断失败的主要原因。采用降低孕妇血清HBVDNA的措施,如对孕妇进行抗HBV治疗,也许能降低HBV宫内感染率。     

单股环DNA病毒基因组的复制及其转录调控因子结合序列

单股环ＤＮＡ病毒基因组的复制及其转录调控因子结合序列崔治中（扬州大学农学院兽医系,生物技术系,扬州２２５００１）关键词单股环ＤＮＡ病毒,基因组复制,调控区结构最近定名的单股环ＤＮＡ病毒科（Ｃｉｒｃｏｖｉｒｉｄａｅ）是迄今为止发现的一类最小的动物病毒。...     

黄芪A_6组分与无环鸟苷联合对Ⅰ型单纯疱疹病毒感染小鼠的治疗作用

黄芪Ａ＿６组分与无环鸟苷联合对Ⅰ型单纯疱疹病毒感染小鼠的治疗作用左丽,董熙昌,孙晓娟（贵阳医学院微牛物学教研室,贵阳５５０００４）关键词黄芪,无环鸟苷,药物协同作用,Ⅰ型中纯疱疹病毒,小鼠单纯疱疹病毒（ＨＳＶ）是人类最常见的病原之一,可引起多种疱疹性...     

乙型肝炎病毒复制水平对原发性肝癌发病的影响

目的:探讨乙型肝炎病毒(HBV)复制水平对原发性肝细胞肝癌(HCC)发病的影响.方法:调查226例HCC患者和51例乙型肝炎后肝硬化(LC)患者,分别应用ELISA法和聚合酶链式反应(PCR)检测血清乙型肝炎病毒标志物(HBV-M)和DNA含量.结果:HCC患者中HBsAg阳性率为96.9%;168例HCC患者和51乙型肝炎后LC患者接受HBV DNA定量检测.阳性率分别为85.1%、88.2%,两组患者lg HBV DNA均服从正态分布,HBV DNA的均数为105.49±1.49拷贝/ml、106.15±1.38拷贝/ml,乙型肝炎后LC组患者血清HBV DNA含量较高(P＜0.05);乙型肝炎后LC患者中HBeAg阳性率较HCC组高(P＜0.05);HCC患者血清HBVDNA含量与HBeAg阳性没有明显的相关性(P＞0.05),乙型肝炎后LC患者血清HBV DNA含量与HBeAg阳性密切相关(P＜0.05);两组患者血清HBV DNA含量与性别、年龄、感染HBV的时间等因素均无明显的相关性(均为P＞0.05).结论:我国HCC的发病与HBV感染密切相关,但可能与患者是否存在HBV高水平复制无关.     

鸭乙型肝炎病毒复制复合体（DHBVRCs）的提纯及其对外源模板的利用

鸭乙型肝炎病毒复制复合体（ＤＨＢＶＲＣｓ）的提纯及其对外源模板的利用邵兴无,陶佩珍,郭巨涛,陈鸿珊（中国医学科学院医药生物技术研究所,北京１０００５０）关键词鸭乙型肝炎病毒复制复合体,ＤＮＡ聚合酶,核心抗原,外源模板１鸭乙型肝炎病毒复制复合体（ＤＨＢ...     

应用微环DNA技术体外诱导和制备重组的乙型肝炎病毒共价闭合环状DNA

乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)是病毒慢性感染的分子基础。本课题组前期研究通过Cre/loxP介导的位点特异性DNA重组策略,在细胞核内由前体质粒诱导重组cccDNA(rcccDNA^loxP)产生,首次建立了HBV cccDNA的体外培养细胞和小鼠实验模型。本研究基于大肠埃希菌ZYCY10P3S2T PhiC31重组酶诱导表达系统,建立了一种体外诱导HBV rcccDNA(rcccDNA^attR)微环产生和纯化的策略。纯化的rcccDNA^attR微环具有超螺旋结构,细胞培养实验证实其能支持功能性的HBV复制和抗原表达。与普通的线性HBV复制子编码质粒相比,rcccDNA^attR尾静脉高压注射小鼠模型能诱导显著延长的病毒抗原血症。因此,本研究在原核表达系统和实验小鼠水平提供了一种更为简化的HBV cccDNA实验模型系统,并再次显示rcccDNA具有显著的稳定性,能作为一种基本策略在小鼠模型中诱导病毒持续感染。     

表达乙型肝炎病毒表面抗原的非复制型重组痘苗病毒疫苗株RVJ123ΔCK的构建及其生物学性状鉴定

利用重组质粒 pNeo-CK与 pNeo-CKLacZ和表达乙型肝炎 (乙肝 )病毒S抗原的重组痘苗病毒RVJ12 3[1] ,构建了非复制型重组痘苗病毒RVJ12 3ΔCK。Southernblot证实 ,非复制型重组病毒RVJ12 3ΔCK基因组C和K片段间与宿主范围和毒力相关的基因稳定缺失 ,同时 ,J片段中插入的乙肝S抗原基因稳定存在。重组病毒RVJ12 3ΔCK在鸡胚成纤维母细胞中可良好繁殖 ,而在人源细胞系中不繁殖或仅低度繁殖 ,但都能表达HBsAg ,并且在病毒一个复制周期内 ,复制型和非复制型病毒HBsAg表达水平无明显差别。     

人白细胞干扰素两种不同制剂对慢性乙型肝炎抗病毒效应的对比观察

本文报道了人白细胞干扰素两种不同制剂对44例慢性乙肝近,远期抗病毒效应的对比观察结果,其中Ⅰ组23例,Ⅱ组21例,Ⅰ组剂量为1×10~6u/次,im每周3次,共四周后改为1×10~6u/次,每周2次,共6周,总计10周为一疗程,Ⅱ组6×10~4u/次im,每周3次,6个月一个疗程,治毕近期效应在HBeAg,HBcAg,DNA-P及HBV-DNA阴转率及总有效率方面,Ⅰ组分别为52.1％,30％,0％,7.1％及52.1％;Ⅱ组分别为72.2％,50％,60％,28.5％及68.3％。治后进行半年以上追踪观察,Ⅰ组20例,Ⅱ组21例,远期效应,在HBeAg,HBcAg,DNAP,HBV-DNA,阴转率及总有效率方面,Ⅰ组分别为40％,33.3%,66.6％,33.3％及40％;Ⅱ组分别为70％,66.6％,80%,60％及71.4％,经统计学处理,两组近、远期HBV复制指标阴转率及总有效率均无显著差异(HBeAg除外),Ⅰ组抗病毒效应并不优于Ⅱ组。扼要讨论两组疗效相近的因素,认为Ⅰ组适当调整剂量及疗程有可能提高疗效,并讨论了治程中肝功能改变的意义,两组均未出现明显的副反应,是较安全的。     

病毒唑及其与干扰素联合对慢性乙型肝炎抗病毒效应观察

本文报道了病毒唑及其与干扰素联合对23例慢乙肝近、远期抗病毒效应观察结果,并与12例未经抗病毒治疗的慢乙肝作对照(Ⅲ组),其中病毒唑与干扰素联合组(I组)12例,病毒唑组(Ⅱ组)11例。近期HBeAg、DNAP及HBV·DNA阴转率,I组分别为(41.6％、55.5％及28.s％,Ⅱ组45.4％、100％及33.3％,Ⅲ组8.3％、16.6％及8.3％。远期HBeAg及DNAP阴转率在I组分别为70％、83.7％。Ⅱ组60％、100％,Ⅲ组8.3％,50％。综合抗病毒效应,近期有效率在I、Ⅱ、Ⅲ组分别为50％、72.7％及16.6％。远期则分别为70％、55.5％及8.3％。无一例出现毒副反应。均完成疗程。上述结果提示病毒唑及其与干扰素联合对慢乙肝均有一定抗病毒效应,联合应用并不优于单一用药,但毒副反应也未见加重,而且病毒唑药源较方便,使用较安全,值得进一步研究。     

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.     

Clonal Expansion of Normal-Appearing Human Hepatocytes during Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections.     

Feedback Circuits in Hepatitis B Virus Infection

A simplified model using kinetic logic is proposed to approach the problem after Hepatitis B viral (HBV) infection. It accounts for several stable regimes or attractors corresponding to the essential dynamic behaviour of the replication of the Hepatitis B virus. Infection with the virus can result in viral clearance, fulminant hepatic failure and death, or chronic transmissible infection, that is multistationarity corresponding to the existence of the positive feedback circuit in our modelling. Another implication of this model is the existence of oscillations or homeostatic mechanisms, sometimes observed in the viral cycle, consistent with the existence of the negative feedback circuit. Thus, this report shows how a simple model of kinetic logic may be used to account for the variety of manifestations of HBV infection. This model implies the presence of the Hepatitis B e antigen, whose conservation suggests that it plays an important role in the life cycle of hepadnaviruses. Its function in the viral cycle is still unknown, but our model suggests that this antigen could explain the passage from one state of the viral infection (acute or latent) to another, as well as the oscillatory behavior which may account for the intermittent symptoms of hepatitis observed in some patients. Furthermore, this model shows a virgin state. This state is also reached after recovery. The model proposed demonstrates that starting from a viral acute infection, the host's immune response, depending on the immunological status of the patient, can lead to viral clearance, or to periodic spontaneous reactivation.     

小剂量干扰素与胸腺肽联合对慢性乙型肝炎抗病毒效应的追踪观察

本文报道小剂量干扰素与胸腺肽联合对慢性乙肝20例抗病毒效应的追踪观察结果,并与同期同批同剂量单纯干扰素治疗13例慢性乙肝作对照,追踪时间均为治后半年—2年,从HBeAg、HBcAg、DNAP、HBV DNA阴转率来看,治疗组分别为58.8％、60％、60％及66.6％、对照组分别为50％、50％、100％及50％。再从HBV四项复制指标改变来看,则治疗组4例全阴转,7例仅一项阳性,总有效率达61.1％(11/18),而对照组仅为20％(2/10),P<0.01,认为干扰素与胸腺肽联合治疗优于对照组,并扼要讨论增强干扰素抗病毒效应的各种措施,认为干扰素与胸腺肽联合,不论从前已报道近期疗效和本文远期追踪来看均较安全而有效,二者合用起到增强抗病毒效应的作用,值得进一步探索。