Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-gamma, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-gamma at 0.1 to 5 microg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5 microg/L, IFN-gamma also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-gamma showed no additive effect, sequential treatment first with lamivudine and then IFN-gamma was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 micromol/L lamivudine for two days, followed by 1 microg/L IFN-gamma for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 micromol/L lamivudine for two days, followed by 5 microg/L IFN-gamma for six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-gamma and lamivudine, especially in IFN-alpha non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.     

Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ

Immune tolerance of human cells without generalized immunosuppression was created in groups of normal fetal rats at 17 days of gestation by inoculation ip with primary human hepatocytes in utero. One day after birth, suspensions of human hepatocytes were transplanted via intrasplenic injection and one week later groups of rats were inoculated with hepatitis B virus (HBV). Tolerized rats that were transplanted with human hepatocytes and subsequently infected with HBV produced hepatitis B surface antigen (HBsAg) in serum beginning on day 3. Levels rose fivefold and remained stable at 0.75 pg/ml through at least 60 days. Of cells that stained positive for human serum albumin, approximately 30% were found to be also positive for HBsAg by immunohistochemistry. Serum HBV DNA was detectable from 1 to 15 weeks postinfection. Finally, covalently closed circular DNA, reflecting HBV replication, was found in liver and serum. Controls that were tolerized and not transplanted, but inoculated with HBV, as well as untreated controls, had no evidence of HBV gene expression or replication under identical conditions. The data support the conclusion that primary human hepatocytes transplanted into genetically immunocompetent rodent hosts, survive and maintain sufficient differentiation to produce human serum albumin and be infected by HBV.     

HBV DNA转染原代鸭肝细胞初步观察

目的：通过观测人HBV DNA在非哺乳动物－－鸭肝细胞中的复制和表达水平，探讨人HBV感染与复制的跨种属特异性，为建立人HBV DNA转染跨种属肝细胞模型奠定基础。方法：获取线性HBV DNA并电转染原代鸭肝细胞，电转后48h采用IMX系统检测鸭肝细胞中HBsAg表达水平，用Southern blot-ting和dot blotting检测HBV DNA复制情况。以单纯电击肝细胞为对照。结果：转染组原代鸭肝细胞裂解液中HBsAg为9．10（P／N值≥2．1为阳性），HBeAg为阴性；上清液中二者均为阴性。转染组原代鸭肝细胞裂解液dot blotting呈强阳性；转染组肝细胞总DNA Southern blotting显示约4.0kb以下分子涂抹带，为游离复制型HBV DNA，包括rcDNA,cccDNA与ssDNA等复制中间体，未见整合型HBV DNA－－高分子区（4.0-24.0kb)涂抹带，对照上述指标均为阴性。结论：人HBV DNA能在原代鸭肝细胞中复制和表达，可能为肝细胞内环境依赖性，无严格种属特异性限制。     

乙型肝炎病毒裸DNA转染原代大鼠肝细胞模型的建立

目的建立乙型肝炎病毒(HBV)环化裸DNA转染原代大鼠肝细胞瞬时表达模型,为进一步研究肝细胞与HBV之间的互动关系奠定基础. 方法采用原代大鼠肝细胞(PRH)作为靶细胞,电转环化HBV DNA,于转染后1～10d各时点分别以Southern杂交和斑点杂交分析HBV DNA的复制中间体与复制形式,以IMX系统检测HBsAg、HBeAg,以western免疫印迹、免疫斑点印迹和免疫细胞化学法检测HBcAg,用RT-PCR检测HBV S/X mRNA,并采用电镜观察有无Dane颗粒合成.以单纯电击PRH为对照组. 结果 HBV环化裸DNA在PRH中的复制为游离型,可见rcDNA、cccDNA和ssDNA等复制中间体;表达的蛋白质产物、HBsAg于转染后各时点PRH裂解液中均可检测到(P/N值4.83～85.69,阳性≥2.1),峰值于1～3d,转染后1～10d平均P/N值为18.239±27.459;PRH培养上清液中未检测到HBsAg;HBeAg于PRH培养上清液和细胞裂解液中均呈阴性(P/N值＜2.1);HBcAg于转染后1～3d时点内检测到低度表达;HBV S mRNA为阳性,而X mRNA为阴性;电镜未观察到Dane颗粒. 结论 HBV环化裸DNA转染原代大鼠肝细胞瞬时表达模型稳定性、可重复性均良好.     

原代培养人胎肝细胞体外感染HBV的研究

目的建立HBV感染人胎肝细胞体外培养系统。方法 首先分离、培养人胎肝细胞，然后应用HBV阳性血清感染体外培养的人胎肝细胞；每隔2d收集上清液和肝细胞，应用ELISA,免疫组化法、原位杂交法和斑点杂交法检测上清液和细胞中HBsAg和HBV DNA。结果 上清液中HBsAg在感染后2d～20d均可测出，以感染后4d～16d达高峰(A值在0．22左右)。免疫组化检测细胞中HBsAg呈阳性表达，原位杂交和斑点杂交检测细胞和上清液中HBV DNA也呈阳性表达。结论 HBV在原代培养人胎肝细胞中能稳定复制和表达至少达16d。     

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

AIM： To explore whether acute cellular DNA damage response is induced upon hepatitis B virus （HBV） infection and the effects of the HBV infection. METHODS： We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chkl phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet （UV） radiation and ionizing radiation （IR）-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS： We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chkl, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h aEer HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increasedapoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION： HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.     

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。     

Dynamics of plasma hepatitis B virus levels after highly active antiretroviral therapy in patients with HIV infection

BACKGROUND/AIMS: The optimal strategy to prescribe highly active antiretroviral therapy (HAART) in patients infected with both hepatitis B virus (HBV) and human immunodeficiency virus (HIV) remains unsettled. This study aimed to compare the HBV dynamics between HBeAg-positive and HBeAg-negative coinfected patients treated with lamivudine-containing HAART. METHODS: We retrospectively analyzed the serial changes of plasma HBV DNA levels in 24 HBsAg-positive HIV-infected patients who entered the HAART program. A polymerase chain reaction-based assay, capable of quantifying as few as 400 HBV copies/ml, was used. The median follow-up time was 18 months. RESULTS: HAART containing lamivudine 300 mg/day effectively suppressed plasma HBV-DNA to 10(-3)-10(-5)-fold of the baseline levels, but a multi-phasic decay of HBV DNA was observed. The later phases became flat, as a persistent residual HBV viremia, in eight of the studied 10 HBeAg-positive patients; in contrast, residual HBV viremia was not observed in the 10 HBeAg-negative patients studied (8/10 vs. 0/10, P=0.0007, Fisher's exact test). HAART without lamivudine did not suppress plasma HBV DNA levels in the remaining four patients. CONCLUSIONS: HAART containing lamivudine 300 mg/day effectively suppress HBV replication in HBeAg-negative HIV/HBV-coinfected patients. Nevertheless, residual HBV replication persisted in most HBeAg-positive coinfected patients.     

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs). METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012 copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group. RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group. CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.     

Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

AIMTo investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODSThe human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTSA stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSIONHBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.     

人乙型肝炎病毒感染与原发性肝癌──—在树忽的实验研究

用人乙肝病毒（ＨＢＶ）接种树的，建立ＨＢＶ感染动物模型，实现连续传代感染，并用乙肝疫苗预防其感染。以此模型研究ＨＢＶ及（或）黄曲霉毒素Ｂ１（ＡＦＢ１）在诱发肝癌中的作用。结果肝癌诱发率在同时接受两种因子者显著高于单一因子者，癌前病变的发生与肝癌诱发率相关，被感染树的肝组织及（或）肝癌中检出ＨＢＶＤＮＡ可整合于宿主肝ＤＮＡ。提示ＨＢＶ和ＡＦＢ１起协同致癌作用并支持ＨＢＶ与肝癌的病因学关系。     

乙型肝炎病毒在异种动物原代肝细胞中复制与表达的研究

目的 探讨乙型肝炎病毒(HBV)DNA复制和表达的跨种属特异性。 方法 分离培养原代大鼠肝细胞(PRH)与原代鸭肝细胞(PDH),电转HBV线性裸DNA(转染组每1×107PRH或PDH 1.19×1012拷贝),分别于转染后1～15d各时点,收集PRH或PDH培养上清液与细胞裂解液,分别以Southern杂交分析和斑点杂交法分析HBV DNA的复制中间体与复制型式;以全自动免疫荧光检测系统检测乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg),以western免疫印迹、免疫斑点印迹和免疫细胞化学法检测乙型肝炎核心抗原(HBcAg);用逆转录聚合酶链反应检测HBV S/X mRNA。以单纯电击PRH/PDH为对照组。 结果 Southern杂交分析显示:PRH/PDH转染组HBV DNA均为游离复制型,可见4.0 kb以下分子区带,包括松弛环状DNA、共价闭环形DNA和单链线形DNA等复制中间体;未见整合型HBVDNA-高分子(4.0～24.0 kb)区带。HBV DNA在PRH中表达蛋白产物水平,HBsAg于转染后各时点PRH裂解液中均可检测到(P/N值2.17～93.41,平均值为14.74±31.82,阳性≥2.1),峰值于1～3 d;仅转染后1d PRH培养上清液中检测到HBsAg,P/N值为6.66;HBcAg和HBeAg仅于转染后1～3 d时点内检测到低度表达;HBV S mRNA为阳性,而X mRNA为阴性。HBV DNA转染PDH组,转染后1、3、5 d各时点PDH裂解液中HBsAg分别为15.24、     

系统检测慢性乙型肝炎患者外周血淋巴细胞免疫表型的临床意义

目的:系统研究慢性乙型肝炎患者外周血淋巴细胞免疫表型以及与临床的关系.方法:用流式细胞术测定28例慢性乙型肝炎患者外周血单个核细胞膜CD3,CD4,CD5,CD25,CD28及CD38等相关CD分子的表达情况,并与22例健康对照组比较,并分析其与HBVDNA及临床的相关性.结果:28例慢性乙型肝炎患者外周血中CD4 CD25 ,CD8 HLADR CD38 ,CD3-CD19 ,CD5-CD19 及CD19 CD38 淋巴细胞数明显高于健康对照组(t=2.37,3.71,4.10,2.31,2.17,P<0.05),而CD3-CD8 ,CD8 CD28-及CD3-CD(16 56) 淋巴细胞数明显低于健康对照组(t=3.14,3.20,2.51,P<0.05).外周血中HBVDNA含量>109copies/L的患者其CD3 CD8 ,CD8 CD28 ,CD4 CD45RA CD62L ,CD8 CD45RA CD62L 及CD4 CD38 淋巴细胞数均显著高于HBVDNA含量<109copies/L的患者,差别有统计学意义(t=2.16,2.42,2.83,3.01,2.50,P<0.01或P<0.05).结论:慢性乙型肝炎患者T、B两种淋巴细胞的活化程度均较高,NK细胞数量减少;体内HBV复制活跃的患者其T淋巴细胞活化程度较高.     

一种新的免疫活性细胞(IPCs)在慢性乙型肝炎患者中的检测

目的近年来研究发现了一种在人的外周血中专职产生α/β干扰素的免疫活性细胞,即“干扰素产生细胞(IPCs)。IPCs在外周血中产生干扰素的量是其他产生干扰素细胞的200～1000倍。因此可以说IPCs是体内IFN的专职产生细胞。 IPCs在慢性乙型肝炎患者体内数量的多少和功能的强弱,决定着患者体内IFN的产量,并直接影响着HBV的清除和病程的转归。本文初步检测了干扰素产生细胞(IPCs)在慢性乙型肝炎患者体内的变化特点,以期进一步明确HBV持续感染的患者中是否存在着IPCs数量和功能的缺失及其对慢性肝炎发病机制的影响。方法随机选取了解放军第三○二医院2001年7月～8月住院的25例慢性乙型肝炎患者,男性19例,女性6例,年龄12～49岁。对照组14例为健康供血员。新鲜分离的抗凝外周全血3ml,用PBS等倍稀释,混合均匀后轻轻加在淋巴细胞分离液面上,血液与淋巴细胞分离液的体积比为2:3,2 500rpm离心25min,轻轻吸界面细胞到一干净离心管中,加PBS(含2％胎牛血清和0.5mMEDTA)悬浮细胞,以1500rpm、1 000rpm离心洗涤细胞2次,洗尽血小板。1×10~6PBMC用荧光标记鼠抗人CD_4-FITC,CD3,CD14,CD16,CD20和CD11c—PE单抗染色25min,1％的多聚甲醛固定,上流式细胞仪检测。按照国外Liu YJ(Blood2001,98(4):906-912)所采用的方法,在前向角和侧向角散点图中找出所有可见的PBMC,先设门R1,记数10~5个PBMC。再以PE标记的CD3,CD14,CD16,CD20,CD11c为横轴,以FITC标记的CD4为纵轴作散点图,设门2,门2内的细胞即我们所要检测的IPCs。记数门2内所有的细胞所占10~5个PBMC的百分数,然后比较慢性乙型肝炎患者和健康人外周血中IPCs数量的变化。结果 25例慢性乙型肝炎患者外周血中IPCs的百分数为0.093±0.078,较正常人(0.326±0.092)明显降低,两者相差3.5倍,有显著差异(P<0.001)。同时发现在HBV DNA(+)的患者中其IPCs的百分数为0.081±0.054,明显高于HBV DNA(-)患者(0.040±0.031),差异显著(P<0.01)。IPCs数量与慢性乙型肝炎患者ALT水平无相关性。结论本文通过检测25例慢性乙型肝炎患者和14例正常人外周血IPCs的数量发现,在慢性肝炎患者外周血中存在着明显的IPCs数量的降低,平均只占其外周血PBMC的0.093％,而明显低于正常人的0.326％(P<0.001)。因此,在临床中我们观察到慢性肝炎患者体内HBV病毒总是在潜伏或增殖,导致病情迁延反复以及在实验研究中发现患者体内IFN的量低于正常人等现象,这些现象最直接的原因可能是其体内IPCs数量的下降所致。同时我们也观察到,虽然在慢性乙型肝炎患者体内存在着IPCs数量的降低,机体的免疫反应受到抑制,但并不是机体处于无反应状态。因为在有HBV复制的病例中,其IPCs为0.081±0.054,与无HBV复制的病例组相比(0.040±0.031),P<0.05。由此可见在病毒的刺激作用下,机体自身的细胞免疫应答也是增强的,但可能由于数量上的不足而不足以抑制病毒的复制,因而HBV DNA呈阳性。本研究只是初步检测了慢性肝炎患者IPCs的数量及其临床意义,有关IPCs的研究还存在着许多待阐明的问题,例如IPCs数量和T细胞亚群变化之间的关系;IPCs水平的高低与患者HBV病毒载量动态变化以及IPCs与慢性肝炎病程之间的关系等问题,有待我们去进一步探讨。     

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with In vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log 10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.     

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM： To establish a cell model harboring replicative clinical hepatitis B virus （HBV） isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B （CHB） patients.
METHODS： The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction （PCR）. All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel.
RESULTS： A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent.
CONCLUSION： The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.     

体内与转染细胞中乙型肝炎病毒株复制特性的相关性

目的 比较不同乙型肝炎病毒（HBV）株在体内及转染细胞中复制特性是否相符。方法 以核酸杂交定量和聚合酶链反应-酶联免疫吸附试验（PCR-ELISA）测定5例孕妇血清中HBVDNA含量,并测定乙型肝炎表面抗原（HBsAg)和乙型肝炎e抗原（HBeAg)含量,分别克隆血清中的HBV基因组转染细胞,检测培养上清液中HBsAg和HBeAg表达水平,并以Southern印迹及核酸杂交检测转染细胞内、外HBVDNA复制水平。结果 转染细胞内、外HBVDNA复制水平与相应血清HBVDNA量呈正相关趋势,转染细胞表达的病毒抗原水平与相应血清中病毒抗原含量也呈正相关趋势。结论 感染者血清中HBVDNA和病毒抗原的含量与毒株在细胞中复制和抗原表达水平有相符趋势。HBV不同毒株在转染细胞中的复制可基本反映体内毒株的复制特性。     

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine.METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization.RESULTS: IFN-γ at 0.1 to 5 μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells.At 5 μg/L, IFN-γ also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γ showed no additive effect,sequential treatment first with lamivudine and then IFN-γ was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 μmol/L lamivudine for two days, followed by 1 μg/L IFN-γ for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 μmol/L lamivudine for two days, followed by 5 μg/L IFN-γ for six days showed a 72% reduction in HBV cccDNA pool.CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γ and lamivudine,especially in IFN-α non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.     

乙型肝炎病毒转基因小鼠免疫耐受状态及血液生物化学指标的研究

目的 了解HBV转基因鼠HBV基因的复制表达和免疫耐受状态,为探讨乙型肝炎发病机制和抗HBV新药评价提供可靠的参考依据.方法 选取遗传背景相同的SPE级HBsAg阴性非转基因鼠和转基因鼠.化学发光法检测HBsAg、HBeAg、HBV DNA,ELISA检测前S1、HBcAg,肝组织行病理学检查,免疫组织化学染色检测不同时期转基因鼠肝HBsAg表达,流式细胞仪检测小鼠淋巴细胞增殖情况,酶联免疫斑点检测(ELISPOT)分泌IFNγ的T淋巴细胞斑点数,双色免疫荧光法检测脾细胞悬液和脾树突状细胞(DC)中Toll样受体(TLR)2和TLR9的表达.数据行t检验和F检验.结果 HBV转基因鼠可复制表达HBsAg、前S1、HBeAg、HBcAg和HBVDNA,而抗-HBs、抗-HBc、抗-HBe均阴性;肝组织无明显病理改变,肝细胞中HBsAg在胞质表达,HBcAg在胞核表达.HBsAg刺激后,HBV转基因鼠T淋巴细胞增殖能力为(697.6±67.3)cpm,显著低于非转基因鼠的(1315.5±191.6)cpm.经HBsAg刺激后,HBV转基因鼠脾细胞分泌IFNγ的T淋巴细胞斑点数为8.25±1.10,低于非转基因鼠的28.50±4.21(F=155.967,P=0.000).HBV转基因DC表达CD11c+、TLR2和TLR9与非转基因鼠比较,差异无统计学意义(均P＞0.05).在18日龄胎鼠和1日龄仔鼠肝组织观察到HBsAg表达.结论 HBV转基因鼠有HBV相关抗原表达,并对HBV相关抗原存在免疫耐受,其先天和获得性免疫功能均正常,类似于人类慢性HBV无症状携带者.HBV转基因鼠是比较理想的动物模型.