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.     

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。     

In vitro replication and expression of hepatitis B virus from chronically infected primary chimpanzee hepatocytes

Primary chimpanzee hepatocytes were maintained in vitro utilizing a serum-free medium. Hepatocyte functions were sustained throughout the culture period as demonstrated by the synthesis and secretion of liver-specific plasma proteins characteristic for differentiated hepatocytes. Hepatocyte cultures established from a chimpanzee chronically infected with human hepatitis B virus exhibited the synthesis and secretion of hepatitis B virus proteins into the medium. In addition, the de novo replication of hepatitis B virus was documented by the recovery of virus, exhibiting an endogenous DNA polymerase activity, from the tissue culture medium. Therefore, both the long-term maintenance of differentiated hepatocytes and the expression of hepatitis B virus from these primary cultures were sustained in the serum-free medium.     

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、     

Gene expression profile of Huh‐7 cells expressing hepatitis C virus genotype 1b or 3a core proteins

Background: The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype. Aim: To identify some hepatitis C virus (HCV) genotype‐specific virus–host interactions potentially leading to clinically significant consequences. Methods: We compared the gene expression profile of Huh‐7 cells transiently expressing the core protein of HCV genotype 1b and 3a using microarray technology. Results: Thirty‐two genes were overexpressed in Huh‐7 transfected with the HCV genotype 1b core protein and 57 genes in cells transfected with the genotype 3a core protein. On the other hand, we found 20 genes downregulated by core 1b and 31 genes by core 3a. These included genes involved in lipid transport and metabolism, cell cycle, immune response and insulin signalling. Conclusion: The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.     

High expression of hepatitis B virus based vector with reporter gene in hepatitis B virus infection system

AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.     

Malignant transformation of the cultured human hepatocytes induced by hepatitis C virus core protein.

BACKGROUND/AIM: Hepatitis C virus core protein (HCV-C) has been known to play an important role in hepatocarcinogenesis. But, up to now there is no certain evidence in pathomorphology directly supporting this standpoint. In this study, a human hepatocytes model expressing HCV-C was established for investigating the influence of HCV-C on hepatocytes biological properties. METHODS: The HCV-C expression plasmid, PcDNA3-C, was transfected into Chang-liver cells to establish HCV-C expressing cells. Proliferation rate and variation index of DNA content of these cells were measured by MTT and FCM. The malignant transformation of these cells was observed by electron microscope. Furthermore, these cells were subcutaneous injected into nude mice to observed their tumor genesis. RESULTS: Proliferation rate and variation index of DNA content of these cells markedly increased. 10/10 of BALB/c-nu/nu nude mice generated tumors at 3 weeks after subcutaneous inoculation of the HCV-C expressing cells. And, histological structure of the tumors coincided with that of hepatocarcinoma. CONCLUSIONS: The HCV-C may play a key role in hepatocarcinogenesis resulting from HCV infection.     

Bile acid formation in primary human hepatocytes

AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.     

HepG2.2.15细胞内乙型肝炎病毒cccDNA的定量检测

目的　建立一种细胞内乙型肝炎病毒cccDNA的定量检测方法。方法　消化收集处于对数生长期的HepG2.2.15细胞,取1×106 个细胞用小量质粒抽提试剂盒抽提细胞内的cccDNA,抽提产物用绿豆核酸酶酶切纯化,所得酶切产物用特异的引物和探针进行选择性荧光定量聚合酶链反应(PCR)检测。用对数生长期前的细胞培养上清液、4 份HBV DNA阳性和2 份HBV DNA阴性的慢性乙型肝炎(轻度)患者血清验证荧光定量PCR法的特异性,并用不同浓度的质粒标准品检测该方法的敏感性。结果　证实HepG2.2.15细胞内存在cccDNA,其含量约为每个细胞18 拷贝。对数生长期前培养上清液和慢性乙型肝炎(轻度)患者血清均未检测到荧光信号,本实验条件下用该方法可检测低至103 拷贝/ml的cccDNA分子。结论　该方法操作方便,特异性高,敏感性较好,可用于定量检测细胞内的cccDNA及抗病毒药物的筛选和评价等。     

乙肝病毒肝源细胞培养模型的研究进展

乙型肝炎病毒感染可以引发慢性病毒性肝炎、肝炎肝硬化,甚至是肝细胞癌。全球约有3.5亿人感染慢性乙型肝炎病毒。随着分子生物学和细胞培养技术在乙肝病毒研究中的发展与应用,研究者们建立起了多种细胞模型。本文简要综述肝源细胞模型在乙型肝炎病毒研究中的应用进展。     

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。     

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.     

Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination

AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.RESULTS: The freshly isolated hepatocytes with high viability (85%) were successfully immortalized using retroviral gene transfer of SV40T. SV40T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.     

An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.

An in vitro culture of human fetal hepatocytes has been employed for infection by hepatitis B virus (HBV) virions that are produced by an established human hepatoma cell line, HB 611. HBV surface antigen and e antigen were released into the medium 3-4 days after infection, and production continued thereafter. RNA synthesis with similar kinetics was observed. Viral DNA replication started 2 days after infection, and replicative HBV DNA that included relaxed circles, single-stranded minus strands, and closed circles accumulated during 16 days of incubation. Immunofluorescent study using fluorescein isothiocyanate-labeled rabbit antisera directed against HBV core antigen revealed that this antigen is present in the nuclei in 12% of the infected cells. Particles containing HBV DNA were detected in the culture medium and were infectious. Thus, this in vitro infection system closely mimics infection in vivo and it allows detailed studies on early events associated with human HBV entry into cells and subsequent replication and integration.     

应用基因芯片技术对乙型肝炎病毒DNA聚合酶反式调节基因的研究

目的应用基因表达谱芯片(基因芯片)技术检测乙型肝炎病毒DNA聚合酶(HBVDNAP)三个功能域[(N末端蛋白(TP),逆转录酶DNA多聚酶(PR),核糖核酸酶H(RNaseH)]的表达对肝母细胞瘤细胞HepG2基因表达谱的影响,进一步阐明HBVDNAP对肝细胞基因表达的调节机制及其生物学功能。方法以常规分子生物学技术分别构建真核表达载体pcDNA3.1()TP,pcDNA3.1()PR和pcDNA3.1()RNaseH,以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析。结果TP有111条基因表达水平上调,88条基因表达水平下调。PR有79条基因表达水平上调,90条基因表达水平下调。RNaseH有113条基因表达水平上调,109条基因表达水平下调。结论应用基因芯片成功筛选HBVDNAP三个功能域蛋白转染细胞后的差异表达基因,为进一步研究HBVDNAP的反式激活作用及免疫调节机制提供了新的依据。