丙型肝炎病毒体外感染胎肝细胞的初步研究

目的：探索原代培养胎肝细胞对丙型肝炎病毒（HCV）的易感性,旨在建立较为稳定实用的细胞感染模型。方法：研究血清与培养肝细胞共同孵育6－8h后,收集不同时相点标本,用逆转录聚合酶链反应（RT－PCR）分别检测细胞内或上清液中正负链RNA,免疫组化检测细胞内HCV NS3,NS5特异性抗原的表达情况,以及原位杂交检测细胞内HCV负链RNA。结果：接种感染血清3d后,即可在细胞内或培养上清液中检出HCV正负链RNA,间断检出至感染后第17天,HCV NS3,NS5特异性抗原可在感染细胞内表达,阳性物质位于乐中,原位杂交法证实细胞内存在负链RNA,也位于胞浆中,结论：原代培养的胎肝细胞对HCV不但易感,而且稳定地支持HCV复制。     

人胎肝细胞体外感染HCV的定性研究

目的：通过定性研究探讨人胎肝细胞在体外与丙型肝炎病毒（ＨＣＶ）阳性血清共培养２４ｈ是否能感染ＨＣＶ。方法：人胎肝细胞在体外与ＨＣＶ阳性血清共培养２４ｈ后,采用原位ＲＴ－ＰＣＲ技术对其进行ＨＣＶ定性检测。结果：通过原位ＲＴ－ＰＣＲ技术检测,发现人胎肝细胞内ＨＣＶ正、负链ＲＮＡ均为阳性。此外,将人胎肝细胞在体外与ＨＣＶ阳性血清共培养２４ｈ并经多次洗涤后继续培养的上清液加入到新鲜分离的正常人胎肝细胞培养     

体外培养妊娠早期人胎肝细胞对乙型肝炎病毒易感性的变化

目的观察妊娠早期(孕10周)人胎肝细胞在不同的体外培养条件下对乙型肝炎病毒(HBV)易感性的变化，为HBV受体的发现和鉴定打下基础。方法设计无血清培养基，分别添加不同的细胞分化诱导物，在不同时相以HBV感染原代培养10周孕龄人胎肝细胞，检测细胞形态、功能及HBV感染标志物。结果培养基中添加2．5mmol／L苯巴比妥钠，培养6d后细胞逐渐出现成熟肝细胞标志，并可被HBV感染；其他培养条件下则不能观察到HBV的成功感染。结论苯巴比妥钠可在体外培养时诱导早期人胎肝细胞分化，并出现HBV易感性。     

HCV HBV感染与肝细胞性肝癌

调查了肝癌高发地区不同肝病患者中丙型肝炎病毒(HCV)感染率。慢性肝病患者绝大多数已被乙型肝炎病毒(HBV)感染。HCV第二代抗体阳性率,肝癌7.3％,肝硬化6.6％,慢性肝炎6.6％和急性肝炎3.4％。两种病毒的复合感染率,肝癌5.1％,肝硬化1.7％,慢性肝炎3.9％和急性肝炎1.1％。在38例HCV抗体阳性的慢性肝病患者中,ALT异常84.2％,有输血史者占57.9％,HCV-RNA阳性率为71.1％。本研究的资料分析提示,在肝癌高发地区尽管HCV抗体阳性率较低,但HCV感染也是肝癌发生的重要病因之一。     

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、     

肝细胞癌组织中HBV、HCV和HGV感染的研究

为了从组织学角度探讨乙型肝炎病毒 (ＨＢＶ)、丙型肝炎病毒 (ＨＣＶ)及庚型肝炎病毒 (ＨＧＶ)与肝细胞癌 (ＨＣＣ)之间的相互关系 ,我们采用免疫组织化学法 ,以抗 ＨＢｓ,抗 ＨＣＶＮＳ5及抗 ＨＧＶＮＳ5 3种单克隆抗体 (单抗 ) ,对 2 6例ＨＣＣ组织中的ＨＢＶ、ＨＣＶ及ＨＧＶ的表达和分布情况进行了研究。材料和方法一、组织来源ＨＣＣ组织均来源于中山医科大学附属肿瘤医院 1998～ 1999年 2 6例原发性肝癌患者手术切除后的石蜡存档标本。其中男 2 4例 ,女 2例 ,年龄 2 5～ 77岁 ,平均年龄 47岁 ,所有标本均经病理确诊。二、免疫组织…     

人肝细胞癌c-myc基因扩增、MTS1/p16基因变异和HBV感染的研究

]目的探讨原癌基因c-myc扩增、抑癌基因MTS1/p16变异以及HBV感染在人肝细胞癌(HCC)发生发展中的作用.方法应用差异PCR结合非变性聚丙烯酰胺凝胶电泳银染及激光扫描(d-PCR-PAGE-Laser)技术检测c-myc基因扩增,应用PCR结合单链构象多态(SSCP)银染法分析MTS1/p16基因变异,PCR检测HBVDNA.结果(1)29例配对肝细胞癌、癌旁组织中c-myc基因扩增阳性率分别为44.83%(13/29)和51.72%(15/29),两者差异无显著性,P＞0.05;但两者均显著高于肝硬化组织c-myc基因扩增的阳性率8.33%(1/12),P＜0.05.(2)只有3例(10.34%,3/29)肝细胞癌中发现MTS1/p16基因纯合性缺失,未发现MTS1/p16基因突变.(3)正常肝脏、肝硬化和肝细胞癌组织中HBVDNA的阳性率分别为14.29%(2/14)、66.67%(8/12)和96.55%(28/29),三者间差异具有高度显著性,P＜0.001,并且HBVDNA的阳性率随肝脏病情的加重而增高(b＝0.3986,P＜0.001).(4)29例肝细胞癌中c-myc基因扩增和HBVDNA存在与否无关(P＜0.01).结论(1)c-myc基因扩增和HBV感染与HCC的发生、发展密切相关,HCC中c-myc基因扩增和HBV感染之间无内在相关性.(2)HCC中MTS1/p16基因纯合性缺失和突变的发生频率较低.[     

丙型肝炎病毒体外感染正常成人肝细胞的初步研究

一、资料与方法 １．正常成人肝组织标本：取意外死亡的３例成年男性,１例成年女性肝脏,均无肝病史,临床生物化学和病理学检查无异常发现,各种肝炎病毒标志物检测均阴性。 ２．采用体外两步灌流法分离正常成人肝细胞。最后用含１０％小牛血清１６４０培养液按细胞密度 １．０× １０６／ｍｌ稀释后接种至 ２５ ｍｌ培养瓶和加爬片的 ２４孔培养板,置于３７℃、 ５％ ＣＯ２培养箱孵育,２－３ｄ更换完全培养液１次,维持培养１月以上。 ３．加适量感染血清至培养瓶（０．２５ｍｌ／瓶）和培养板（０．１ｍｌ／孔）中,混匀,置于 ３７…     

隐匿性乙型肝炎病毒(HBV)感染

乙型肝炎病毒(HBV)感染是最主要的病毒性感染之一。多数感染可无临床症状，依赖于血清病毒标志物(HBVM)检测可明确诊断，其中血清乙型肝炎表面抗原(HBsAg)阳性是HBV感染的重要依据。而HBsAg的阴转及抗．HBs的出现一直认为是HBV清除和临床痊愈的标志。而近年来，随着分子生物学技术在病毒     

丙型肝炎病毒体外可感染树鼩肝细胞

目的 探讨丙型肝炎病毒（HCV）体外感染树目句原代肝细胞。方法 用HCV RNA阳性血清感染原代树鼩肝细胞，并用感染后细胞培养上清液进行传代感染，通过检测受染肝细胞正、负链HCV RNA、培养上清液中包装后HCV RNA，并对比分析感染前后病毒准种变化等指标，评价感染是否成功。结果 受染树鼩肝细胞自第5～10天可检出负链HCV RNA，而正链RNA至感染后第14天仍可检出；感染后3～14d培养上清液中可检出HCV RNA，且呈RNA酶抗性；培养上清液中病毒可传代感染新的树鼩肝细胞；感染前后HCV准种分析显示树鼩肝细胞可被特定的准种选择性感染，而传代感染后肝细胞可检出新的准种。结论 原代树鼩肝细胞体外可被HCV感染且支持病毒复制。     

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.     

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.     

Hepatitis B virus genotypes and G1896A precore mutation in 486 Spanish patients with acute and chronic HBV infection

This study aims to determine the prevalence of hepatitis B virus (HBV) genotypes (A-F) and their association with the G1896A precore mutation in 486 patients positive for HBV surface antigen. Genotypes were determined by RFLP and precore mutation by real-time PCR. Genotypes D (48.1%) and A (39.5%) were the most common, followed by F (4.1%) and B, C and E (<1%). The A to D ratio (A:D) was 1.4 in HBeAg+ chronic hepatitis B (CHB), 0.6 in HBeAg- CHB and 1.4 in HBeAg- inactive carriers. Distribution of these genotypes was different between HBeAg+ CHB and HBeAg- CHB (P = 0.02), and between HBeAg- CHB and HBeAg- inactive carriers (P = 0.009). Genotype A was the most prevalent in HBeAg+ CHB with elevated alanine aminotransferase (ALT) (68.6%) and genotype D in HBeAg+ CHB with fluctuating ALT (60.7%). There was a difference in genotype prevalence between chronic and acute infection (P = 0.03). The precore mutant correlated with high levels of HBV-DNA in genotype d HBeAg- CHB. Genotype D is not as highly prevalent in Spanish patients as would be expected in a Mediterranean area. The unequal prevalence of genotypes between acute and chronic infection suggests that genotype A is associated with a higher tendency to cause chronic infection.     

HBV动物模型研究进展

HBV感染动物模型是研究HBV致病机制、筛选新型有效抗HBV药物和治疗方法的重要工具,然而HBV感染具有高度组织特异性以及种属特异性,这给HBV感染动物模型的建立带来了困难。近年来,随着分子生物学、实验动物学、病毒学及免疫学等相关学科技术的进步,HBV感染或复制动物模型取得了明显进展。目前应用于HBV(包括与HBV具有相似特性的动物肝炎病毒)研究的动物模型主要包括黑猩猩、树鼩、土拨鼠及鸭HBV感染模型,HBV转基因小鼠、高压水动力注射介导的小鼠HBV复制模型和重组腺相关病毒载体介导的小鼠HBV复制模型,以及人源化人-鼠嵌合肝脏HBV感染模型,此外,钠离子-牛磺胆酸共转运蛋白转基因小鼠感染模型是近年的研究热点。本文就上述HBV动物模型的研究进展进行综述。     

Peripheral T-lymphocyte subpopulations in different clinical stages of chronic HBV infection correlate with HBV load

AIM： To characterize the peripheral T-cell subpopulation profiles and their correlation with hepatitis B virus （HBV） replication in different dinical stages of chronic HBV infection.
METHODS： A total of 422 patients with chronic HBV infection were enrolled in this study. The patients were divided into three stages： immune-tolerant stage, immune active stage, and immune-inactive carrier stage. Composition of peripheral T-cell subpopulations was determined by flow cytometry. HBV markers were detected by enzyme-linked immunosorbent assay. Serum HBV DNA load was assessed by quantitative real-time poiymerase chain reaction.
RESULTS： CD8^＋ T-cells were significantly higher in patients at the immune-tolerant stage than in patients at the immune-active and -inactive carrier stages （36.87 ± 7.58 vs 34.37 ± 9.07, 36.87 ± 7.58 vs 28.09 ± 5.64, P 〈 0.001）. The peripheral blood in patients at the immune-tolerant and immune active stages contained more CD8^＋ T-cells than CD4^＋ T-cells （36.87 ± 7.58 vs 30.23 ± 6.35, 34.37 ± 9.07 vs 30.92 ± 7.40, P 〈 0.01）, whereas the peripheral blood in patients at the immune- inactive carrier stage and in normal controls contained less CD8^＋ T-cells than CD4^＋ T-cells （28.09 ± 5.64 vs 36.85 ±6.06, 24.02 ± 4.35 vs 38.94 ± 3.39, P 〈 0.01）. ANOVA linear trend test showed that CD8^＋ T-cells were significantly increased in patients with a high viral load （39.41 ± 7.36, 33.83 ± 7.50, 31.81 ± 5.95 and 26.89 ± 5.71, P 〈 0.001）, while CD4^＋ T-cells were significantly increased in patients with a low HBV DNA load （37.45 ± 6.24, 33.33 ± 5.61, 31.58 ± 6.99 and 27.56 ± 5.49, P 〈 0.001）. Nultiple regression analysis displayed that log copies of HBV DNA still maintained its highly significant coefficients for T-cell subpopulations, and was the strongest predictors for variations in CD3^＋, CD4^＋ and CD8^＋ cells and CD4^＋/CD8^＋ ratio after adjustment for age at HBV-infection, maternal HBV-infection status, presence of hepatitis B e antigen and HBV mutation.
CONCLUSION： Differences in peripheral T-cell subpopulation profiles can be found in different clinical stages of chronic HBV infection. T-cell impairment is significantly associated with HBV load.     

HBsAb阳性隐匿性HBV感染者血清、肝组织HBV全基因序列分析

目的 了解HBsAb阳性隐匿性HBV感染者血清和肝组织中的HBV基因序列,并比较其差异性.方法 以1例长期随访HBsAb阳性隐匿性HBV感染者作为研究对象,用多种试剂盒检测其血清HBsAg、HBsAb,提取外周血血清和肝组织HBV DNA进行全基因组分段扩增,行序列测定及同源性比较.结果 多种试剂盒检测均提示该例患者HBsAg阴性、HBsAb阳性;血清HBV DNA为103～ 105拷贝/mL;血清和肝组织来源的HBV DNA全基因测序完全相同,均为3 215个碱基、B基因型,与参照序列核苷酸同源性为98.82％,各编码区均没有缺失或移码突变,不同编码区的核苷酸序列同源性为98.37％～ 100％,氨基酸序列同源性为98.18％～ 100％,在S区存在几种变异如PreS1的Q80H、S的C64Y、E164G、L175S,但前S区、“a”决定簇、1 762/1 764、1 896位点均未见变异.结论 HBsAb阳性隐匿性HBV感染者血清和肝组织来源的HBV基因序列无明显差异.