复方六月雪体外抗HBV的实验研究

目的观察复方六月雪(CLYX)体外抗乙型肝炎病毒(HBV)的作用。方法在最大无毒浓度(TC0)基础上观察不同浓度药物作用于HepG2.2.15细胞,分别在第4天和8天收集细胞培养上清液,采用实时荧光定量PCR法检测上清液HBV DNA的含量,采用ELISA法测定上清液HBsAg和HBeAg的滴度。结果无毒浓度的复方六月雪在HepG2.2.15细胞培养中可有效地抑制细胞HBV DNA的复制及HBsAg和HBeAg的分泌。结论CLYX在体外有显著的抗HBV的作用。     

愈肝胶囊对2.2.15细胞分泌HBsAg、HBeAg的抑制作用

目的：观察愈肝胶囊对HBV转染细胞表达功能的影响。方法：本研究以乙型肝炎病毒(HBV)基因转染的人肝癌细胞系2．2．15细胞作为筛选抗HBV药物的细胞模型，观察愈肝胶囊对2．2.15细胞分泌HBsAg、HBeAg的影响；并以四甲基氮唑蓝(MTT)比色法检测药物对细胞的毒性。结果：愈肝胶囊对2．2．15细胞分泌HBsAg和HBeAg有显著抑制作用。半数中毒剂量(TC50)为1．79mg／ml，对2．2．15细胞分泌HBsAg和HBeAg抑制率分别为70．81％，83．96％；半数有效剂量(IC50)HBsAg为0．86，HBeAg为0．13。治疗指数(TI)分别为HBsAg2．08，HBeAg13．76。结论：愈肝胶囊在体外有一定的抗HBV作用。     

慢性乙型肝炎肝组织内HBsAg、HBcAg的表达及临床研究进展

一直以来临床将血清乙型肝炎e抗原(HBeAg)、乙肝病毒DNA(HBV DNA)阳性作为乙肝病毒复制的标志,随着肝穿活检及抗病毒治疗的研究进展,肝活检组织中乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)的表达模式与血清乙型肝炎病毒(HBV)DNA定量、肝组织炎症活动度分级及纤维化分期之间关系的临床研究日益增多,本文就HBsAg和HBcAg在肝组织的表达模式及临床研究进展综述如下.     

壮药汗衣台体外抗乙型肝炎病毒的疗效

目的:研究汗衣台体外抗乙型肝炎病毒(hepatitis B virus,HBV)的疗效.方法:不同浓度汗衣台与体外培养的2.2.15细胞共同孵育,以XTT方法检测其对2.2.15细胞的不良反应,以此决定汗衣台的安全浓度范围;3d更换一次含药培养液,9d后收集上清液;用ELISA法测定上清液中HBsAg和HBeAg含量,荧光定量PCR法检测上清液中HBV DNA的分泌.结果:各浓度汗衣台对HBsAg、HBeAg均有明显抑制作用,抑制率呈剂量依赖性:500-800μg/mL汗衣台对HBsAg的抑制明显高于同浓度的拉米夫定(P=0.002-0.000)及干扰素(P=0.006-0.003),100-800μg/mL浓度汗衣台对HBeAg的抑制明显高于同浓度拉米夫定(P=0.002-0.000)及干扰素(P=0.003-0.002),差异均有显著性.各浓度汗衣台对HBV DNA抑制亦呈剂量依赖性,500-800μg/mL浓度汗衣台短期内抑制HBV DNA分泌的疗效优于同浓度干扰素(P=0.018-0.031),但不如拉米夫定.结论:壮药汗衣台具有一定的体外抗HBV作用.     

叶下珠对乙型肝炎病毒结构与复制的影响

采用叶下珠(Phyllanthus,urinaria)乙醇提取物进行了药物对乙型肝炎病毒(HBV)抗原抗体结合抑制试验、病毒DNA聚合酶抑制试验以及在2.2.15细胞培养中对乙肝病毒表面抗原(HBsAg)和e抗原(HBeAg)表达抑制试验和DNA复制抑制试验,结果叶下珠提取物对上述指标均有不同程度的抑制作用,提示叶下珠为一潜在的抗HBV药物。     

左归丸提取液对2.2.15细胞乙肝病毒标志物的影响

目的:研究左归丸提取液对乙型肝炎病毒e抗原(HBeAg)、表面抗原(HBsAg)的抑制作用。方法:应用2.2.15细胞株作为体外抗乙型肝炎病毒(HBV)实验模型,通过细胞毒性实验以确定对细胞无毒性作用的最高药物浓度,将此浓度药物直接加到细胞培养液中,分别取第4天、第8天的细胞培养上清液,用固相放射免疫方法测定其HBsAg、HBeAg含量。结果:浓度为25、12.5、6.25、3.125mg/ml的左归丸提取液对HBsAg、HBeAg有明显抑制作用;各浓度间存在量效关系;对HBeAg的作用优于对HBsAg的作用。结论:左归丸提取液对2.2.15细胞HBV标志物有显著的抑制作用。     

氧化苦参碱抗乙型肝炎病毒的体外实验研究

目的：观察氧化苦参碱体外抗乙型肝炎病毒（HBV）的活性。方法：以HepG2－2．2．15细胞株为模型, 用微粒酶免疫测定技术(MEIA）检测细胞培养上清液中HBsAg、HBeAg含量,用bDNA信号可扩增法定量检测细胞内核心颗粒HBV DNA的变化,以MTT比色法观察药物的细胞毒性。结果：氧化苦参碱2000μg/ml时对HBsAg、HBeAg的抑制率分别达40．57％、48．27％;浓度为100－200μg/ml时,能明显降低2．2．15细胞浆核心颗粒HBV DNA水平;无明显细胞毒性作用。结论：体外细胞培养表明,氧化苦参碱具有直接抗HBV活性。     

扇贝多糖体外抗乙型肝炎病毒活性的研究

目的体外观察扇贝多糖抗乙型肝炎病毒（HBV）的作用。方法采用HepG2.2.15细胞模型进行体外培养,给予不同浓度扇贝多糖,作用9d后收集上清液,用MTT法观察扇贝多糖对HepG2.2.15细胞的抑制作用;用ELISA法测定上清液中HBsAg和HBeA含量;用荧光定量PCR检测扇贝多糖对HepG2.2.15细胞分泌HBV DNA的影响。结果扇贝多糖浓度在500μg/mL 时,对细胞无明显毒性;扇贝多糖对HBsAg、HBeAg均有抑制作用,其半数有效浓度（IC50）分别为294μg/mL 、168μg/mL,治疗指数（TI）为>17.0和>29.8;扇贝多糖对HBV DNA的复制也有一定的抑制作用。结论扇贝多糖具有一定的体外抗HBV作用。     

核苷的亲脂性磷酸酰胺酯β-LPA体外抗乙型肝炎病毒的作用

目的:探讨β-LPA体外抗乙型肝炎病毒(hepatitis B virus,HBV)的作用. 方法:通过β-LPA干预培养HepG2.2.2.15细胞,ELISA法检测上清HBsAg和HBeAg,32p标记HBV DNA为探针,Southern blot法检测细 胞内的HBV DNA,再以计算机图像处理进行定量分析,得出50%抑制的药物浓度(ED50),以MTT法检测不同浓度药物的细胞毒性,求出50%细胞抑制的药物浓度(ID50).结果:β-LPA体外明显抑制HBV DNA的复制,并呈浓度依赖性.ED50为0.01 μmol/L,β-LPA 细胞毒性实验显示ID50为50 μmol/L.低浓度的β-LPA对上清HBsAg,HBeAg无明显影响,高浓度时有显著的抑制作用.结论:β-LPA具有明显的体外抑制病毒DNA复制作用,且细胞毒性小.     

肝舒胶囊在体外细胞培养中抗HBV活性的研究

目的研究肝舒胶囊在2.2.15细胞培养中抗乙型肝炎病毒(HBV)作用。方法用2.2.15细胞体外培养.对肝舒胶囊抗HBV活性进行评价。结果肝舒胶囊5g／L加药后4d对2.2.15细胞HBsAg和HBeAg的抑制率为51.16％和74.83％,在加药后第8天抑制作用达高峰,对HBsAg和HBeAg的抑制率分别为75.72％和80.02％。药物作用12d时．其治疗指数可达7.73和5.39。结论肝舒胶囊在体外细胞培养中对HBsAg和HBeAg的分泌均有较好的抑制作用。     

青蒿琥酯对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响

目的 探讨青蒿琥酯在体外对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响.方法 将不同浓度青蒿琥酯作用于转染乙型肝炎病毒全基因组DNA的肝癌细胞株HepG2.2.15,收集48 h上清,采用酶联免疫吸附实验检测上清中乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg),采用荧光定量PCR法检测HBV-DNA,流式细胞术检测细胞凋亡.结果 青蒿琥酯对HBV复制具有抑制作用,随着浓度增加,对HBsAg和HBeAg的抑制率逐渐上升,细胞内HBV-DNA 复制水平下降;青蒿琥酯可诱导肝癌细胞早期凋亡及导致细胞死亡,随浓度增加,HepG2.2.15细胞早期凋亡率及死亡率均增加.结论 青蒿琥酯对HepG2.2.15细胞HBsAg和HBeAg的分泌及HBV-DNA复制具有抑制作用,并具有诱导HepG2.2.15细胞凋亡的作用.     

Serum hepatitis B virus DNA detects cryptic hepatitis B virus infections in multitransfused hemophilic patients

The recognition of replicating hepatitis B virus (HBV) may be important to both define the cause of and know how to manage chronic liver disease in multitransfused hemophilic patients. Replicating HBV can be detected at the molecular level by methods for HBV-specific DNA (HBV-DNA), which are much more sensitive than the immunologic methods for detecting hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Unselected hemophilic patients (260; 6% with HBsAg, 4% with isolated anti-hepatitis B core (anti-HBc), 52% with anti-HBs and anti-HBc, 26% with isolated anti-HBs, and 12% with no HBV marker) were investigated retrospectively with a dot spot hybridization technique that detects serum HBV-DNA down to 0.5 pg and by Southern blot analysis, which tests the specificity of the HBV-DNA reactions. Eighteen patients (7%; five with serum HBsAg and 13 HBsAg seronegative with antibodies to HBV) had serum HBV-DNA. Serum HBV-DNA was detected more frequently in HBsAg carriers than in seronegative patients (33% versus 6%, P less than .01), and had no relationship to serum alanine aminotransferase. Serum HBV-DNA was more sensitive than the radioimmunoassay for HBeAg was for detecting replicating HBV (7% versus 1.1%, P less than .01). These findings demonstrate that there is cryptic HBV infection in a number of hemophiliacs and that serum HBV-DNA may coexist with markers thought to reflect immunity against HBV.     

Expression of pre-S gene-encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison to other markers of active virus replication

Pre-S gene-encoded proteins of the hepatitis B virus (HBV) were studied in the liver by immunofluorescence and in serum by radioimmunoassay in 30 patients with chronic HBV infection. The results were compared with molecular hybridization analysis of HBV-DNA in liver and serum, with serum hepatitis B e antigen/antibody (HBeAg/anti-HBe) status and with underlying liver histology. Pre-S peptides were detected in the serum of 11 patients, 10 of whom were positive for serum HBV-DNA and/or liver hepatitis B core antigen. Only 4 of these patients were HBeAg positive. The prevalence of serum pre-S among HBV replicating carriers was 59% (10/17) compared to only 8% (1/13) among those with non-replicating virus (P less than 0.01). All patients with circulating pre-S peptides had active liver disease. Anti-pre-S was detected in the serum of only 4 patients, 3 with integrated HBV-DNA. In contrast to serum findings, pre-S peptides were detected in the liver of all patients with histochemically demonstrable hepatitis B surface antigen (HBsAg), regardless of HBV replicative status. HBsAg carriers with integrated HBV-DNA had abundant cytoplasmic pre-S1 and pre-S2 localized in numerous ground-glass hepatocytes. It is concluded that pre-S peptides are usually displayed in the liver simultaneously with histochemically detectable HBsAg; they are secreted in the serum in association with high HBV replication and release of HBV particles, but in the absence of episomal HBV replication, pre-S peptides seem to be largely retained within hepatocytic membranes.     

Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells

The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents.     

Early upsurge in anti-HBs titer possibly caused by the immunomodulative, not by the mutagenetic effect of interferon and ribavirin

A patient with chronic hepatitis B and C undergoing treatment with interferon and ribavirin showed an upsurge in hepatitis B virus surface antibody (anti-HBs) titer, accompanied by a decrease in hepatitis B virus surface antigen (HBsAg) during the early treatment phase. Simultaneously, elevation of alanine aminotransferase (ALT) was observed. Subsequently, the hepatitis B virus (HBV) DNA titer decreased and HBV e antigen (HBeAg) to anti-HBe seroconversion occurred. The anti-HBs titer gradually returned to the pretreatment level after cessation of ribavirin treatment and HBV-DNA became undetectable. We found no nucleotide mutations in HBV-DNA that could explain the sudden elevation in anti-HBs titer. The appearance of anti-HBs was considered to be a break in immune tolerance against some epitopes in HBsAg, possibly the r epitope, stimulated by interferon/ribavirin treatment. The immunomodulatory effect of ribavirin might have caused this unexpected early immune response to HBsAg that preceded seroconversion to anti-HBe.     

Detection of HBeAg/anti-HBe immune complexes in the reactivation of hepatitis B virus replication among anti-HBe chronic carriers

Sixty-four chronic hepatitis B surface antigen (HBsAg) carriers with hepatitis B e antibody (anti-HBe) were followed in order to detect reactivations of hepatitis B virus (HBV) infection and to assess the incidence and specificity of hepatitis B e antigen/hepatitis B e antibody (HBeAg/anti-HBe) immune complexes (ICs). In 18 out of 19 patients who suffered an increase in alanine transaminase (ALT) values, serum HBV-DNA reappeared co-occurring with the peak(s) of transaminases. HBeAg/Anti-HBe immune complexes were detected in 17/18 (94.4%) patients positive for HBV-DNA. In nine of them, the appearance of immune complexes co-occurred with prednisone therapy, in two following seroconversion after recombinant interferon alpha-2A treatment, and spontaneously in the remaining seven patients. When ALT levels dropped to normal values, immune complexes as well as HBV-DNA became undetectable. In conclusion, the detection of HBeAg/anti-HBe immune complexes seems to be a specific method to detect HBV replication among anti-HBe positive patients.     

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。     

Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line

AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregeno...     

HBeAg negative serological status and low viral replication levels characterize chronic hepatitis B virus-infected women at reproductive age in Greece： A one-year prospective single center study

AIM: To evaluate the seroprevalence of hepatitis B surface antigen (HBsAg) in 13 581 women at reproductive age and the hepatitis B e antigen (HBeAg)/anti-HBe status as well as serum hepatitis B virus (HBV)-DNA levels in a subgroup of HBsAg(+) pregnant women at labor in Greece. METHODS: Serological markers were detected using enzyme immunoassays. Serum HBV-DNA was determined by a sensitive quantitative PCR assay. Statistical analysis of data was based on parametric methodology. RESULTS: Overall, 1.156% of women were HBsAg(+) and the majority of them (71.3%) were Albanian. The prevalence of HBsAg was 5.1% in Albanian women, 4.2% in Asian women and 1.14% in women from Eastern European countries. The prevalence of HBsAg in African (0.36%) and Greek women (0.29%) was very low. Only 4.45% of HBsAg (+) women were also HBeAg(+) whereas the vast majority of them were HBeAg(-)/anti-HBe(+). Undetectable levels of viremia (<200 copies/mL) were observed in 32.26% of pregnant women at labor and 29.03% exhibited extremely low levels of viral replication (<400 copies/mL). Only two pregnant women exhibited extremely high serum HBV-DNA levels (>10 000 000 copies/mL), whereas 32.26% exhibited HBV-DNA levels between 1 500 and 40 000 copies/mL. CONCLUSION: The overall prevalence of HBsAg is relatively low among women at reproductive age in Greece but is higher enough among specific populations. The HBeAg(-)/anti-HBe(+) serological status and the extremely low or even undetectable viral replicative status in the majority of HBsAg(+) women of our study population, suggest that only a small proportion of HBsAg(+) women in Greece exhibit a high risk for vertical transmission of the infection.     

Serologic markers of hepatitis B virus (HBV) and hepatitis D virus infection in carriers of hepatitis B surface antigen who are frequently exposed to HBV

Serum hepatitis B e antigen (HBeAg) and HBV DNA are indicators of active replication of HBV, whereas IgM antibody to hepatitis B core antigen (IgM anti-HBc) may indicate an active immune response to chronic HBV infection. Fifty-eight carriers of hepatitis B surface antigen (HBsAg) who had frequent parenteral exposures were studied for the presence of HBeAg, HBV DNA, IgM anti-HBc and hepatitis delta virus (HDV) serologic markers. Active replication of HBV was detected in 36.2% (25% of drug addicts, 16.7% of thalassemia patients, and 46.9% of hemodialysis patients) and seropositivity for IgM anti-HBc in 55.2% of the HBsAg carriers. Among the 39 HBsAg carriers who were negative for HBeAg, IgM anti-HBc was detected significantly more frequently than HBV DNA (46.1% vs. 5.1%, p less than 0.001). Serologic evidence of HDV infection was detected in 35% of drug addicts, 50% of thalassemia patients and in 9.4% of hemodialysis patients. These data revealed that continued replication of HBV was more frequent in hemodialysis patients than in drug addicts and thalassemia patients who are HBsAg carriers and the opposite was true for the prevalence of HDV infection.