Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2

OBJECTIVE: To construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair. METHODS: The eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis. RESULTS: Restriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system. CONCLUSIONS: Genomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.     

Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus

BACKGROUND/AIMS: Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS: Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS: HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS: The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.     

Human microRNA hsa‐miR‐1231 suppresses hepatitis B virus replication by targeting core mRNA

Pathogen‐specific miRNA profiles might reveal potential new avenues for therapy. To identify miRNAs directly associated with hepatitis B virus (HBV) in hepatocytes, we performed a miRNA array analysis using urokinase‐type plasminogen activator (uPA)–severe combined immunodeficiency (SCID) mice where the livers were highly repopulated with human hepatocytes and human immune cells are absent. Mice were inoculated with HBV‐infected patient serum samples. Eight weeks after HBV infection, human hepatocytes were collected from liver tissues, and miRNAs were analysed using the Toray 3D array system. The effect of miRNAs on HBV replication was analysed using HBV‐transfected HepG2 cells. Four miRNAs, hsa‐miR‐486‐3p, hsa‐miR‐1908, hsa‐miR‐675 and hsa‐miR‐1231 were upregulated in mouse and human livers with HBV infection. These miRNAs were associated with immune response pathways such as inflammation mediated by chemokine and cytokine signalling. Of these miRNAs, hsa‐miR‐1231, which showed high homology with HBV core and HBx sequences, was most highly upregulated. In HBV‐transfected HepG2 cells, overexpression of hsa‐miR‐1231 resulted in suppression of HBV replication with HBV core reduction. In conclusion, a novel interaction between hsa‐miR‐1231 and HBV replication was identified. This interaction might be useful in developing new therapeutic strategies against HBV.     

Characterization of microRNA expression profiles associated with hepatitis B virus replication and clearance in vivo and in vitro

Background and Aim: Alpha interferon (IFN‐α) is an approved treatment for chronic hepatitis B (CHB). MicroRNA (miRNA) are currently known as a part of IFN‐mediated antiviral defense. We aimed at characterizing the miRNA expression associated with hepatitis B virus (HBV) replication and IFN‐mediated HBV clearance. Methods: We investigated the expression patterns of cellular miRNA induced by HBV replication and/or IFN‐α treatment in HepG2 cells, and also analyzed the miRNA response in peripheral blood mononuclear cells in CHB patients on IFN‐α treatment. The differentially expressed miRNA were verified using quantitative real‐time polymerase chain reaction and an miRNA expression pattern was classified based on the final virological response. Results: A total of 223 miRNA were differentially expressed (> 1.5 folds) between the HepG2.2.15 and HepG2 cells, including 24 highly differentially expressed miRNA (> 5 folds). With 12 h of IFN‐α treatment, 23 totally differentially expressed miRNA were identified in HepG2 cells; whereas only five miRNA were identified in HepG2.2.15 cells. Similar amounts of the miRNA were regulated in patients with HBeAg or non‐HBeAg seroconversion; whereas levels of eight miRNA were significantly differentially expressed between the two groups. Conclusions: HBV replication alters miRNA expression profiles and impairs IFN‐inducible miRNA response in HepG2 cells. The miRNA expression pattern of peripheral blood mononuclear cells in CHB patients with IFN therapy can be associated with their therapeutic outcome.     

Apoptosis and its pathway in X gene-transfected HepG2 cells

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in-three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2(0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/ pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.     

Establishment of mice model with human viral hepatitis B

AIM:To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphoo/tes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA.METHODS: HBV DNA was obtained from digested pBR3222HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA.BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection.ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5&#215;10^7 per mouse).Forty-eight hours later,the level of serum protein and transaminase was detected with biochemical method,liver and kidney were sectioned and stained by HE to observe the pathological changes.RESULTS: By enzyme digestion with Eco RI, Xho I and Hind Ⅲ,the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome,which was inserted in the positive direction.HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg.After immunized by pcDNA3-HBV for 4 weeks,HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphoo/tes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage.CONCLUSION:A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably.Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV.A mice model of acute hepatitis with HBV has been established.     

表达乙肝病毒YVDD变异株肝癌细胞模型的建立

目的：建立能稳定表达乙肝病毒（HBV）YVDD变异株的细胞模型，为研究HBV YVDD变异株的生物学特性和进行抗病毒药物筛选提供细胞模型。方法：以pcDNA3．1-HBV-YVDD为模板设计两对引物，扩增带不同酶切位点的HBV全基因，扩增产物纯化后，经酶切、连接，得到pcDNA3．1-HBV．YVDD重组体。采用脂质体转染技术，将pcDNA3．1-HBV-YVDD重组体转染体外培养的HepG2细胞。细胞培养上清液中HBsAg和HBeAg的检测用酶联免疫吸附法（ELISA）法，反转录聚合酶链反应（RT-PCR）法检测HBV多聚酶的表达。结果：建立了HBV-YVDD变异株体外感染的细胞模型HepG2-2HBV-YVDD。该细胞模型能表达较高水平的HBsAg和HBeAg，并能检测到多聚酶的表达。结论：成功构建了能较稳定表达HBV-YVDD变异株的细胞模型。     

新型乙型肝炎病毒细胞感染模型的建立

目的:建立一新型细胞株,使该细胞株既能自然感染HBV又能传代培养,评价杂交细胞对HBV的自然感染能力.方法:分离培养未携带HBV的人原代肝细胞,与诱导突变的HepG2细胞进行融合得杂交细胞,经HAT培养基筛选,利用胰蛋白酶G显带技术鉴定所得细胞,用含HBV的慢性乙型肝炎患者血清感染杂交细胞(同时感染HepG2作为对照),巢式PCR检测感染后细胞内HBVDNA合成及分泌情况及有无HBV复制中间产物HBV cccDNA,间接免疫荧光检测感染后细胞内HBcAg的表达,电化学发光法检测感染后细胞培养上清中的HBsAg和HBeAg.结果:成功建立人原代肝细胞与HepG2的杂交细胞,能体外传代培养,染色体核型分析示杂交细胞染色体众数为99条,证实为融合细胞,HBV感染后第4天起,杂交细胞内和培养上清中均能检测到HBV DNA,HBV感染后第3天起,杂交细胞内可以检测到HBV的复制中间产物HBV cccDNA,HBV感染后第4天起,杂交细胞胞质及部分胞核内HBcAg始终是阳性表达,间接免疫荧光染色呈胞质弥漫性着色,电化学发光法检测培养上清中HBsAg及HBeAg持续表达;而感染后的HepG2细胞检测结果均为阴性.结论:成功建立的兼具有人原代肝细胞和HepG2遗传特性的杂交细胞株可以被慢性乙型肝炎患者血清中的HBV病毒自然感染,可进一步用作研究HBV感染的体外细胞模型.     

维甲酸诱导基因-I诱导干扰素的产生在乙型肝炎病毒感染清除中的作用

目的探讨维甲酸诱导基因-I(RIG-I)在诱导HBV感染IFN产生中的影响,以期对阐明乙型肝炎慢性化机制,寻找新的治疗靶点提供新的思路。方法以RIG-I表达载体flagRIG-l-ful1转染培养6孔板中的HepG2、HepG2.2.15,24h后用水疱性口炎病毒(VSV)感染细胞,然后在0、8、16和24h收集细胞及培养上清液,采用quantitive RT-PCR、Western印迹检测RIG-I、MDA5、IPS-1、ISG54等基因的表达,ELISA检测培养上清液中分泌的IFN-β水平。结果 HepG2.2.15细胞在VSV感染后IFN-β分泌水平(11.18±1.34)pg/mL明显低于HepG2细胞(275.50±22.97)pg/mL(P<0.01);通过质粒flagRIG-I-ful1转染高表达RIG-I后,HepG2.2.15分泌IFN-β的能力恢复至(548.78±57.99)pg/mL与HepG2细胞(532.10±39.34)pg/mL接近的水平(P=0.7013)。结论 HepG2.2.15细胞感染VSV后IFN产生障碍,高表达RIG-I后IFN表达水平得到恢复,提示RIG-I功能缺陷导致抗病毒免疫应答降低,RIG-I可能在HBV感染清除中起重要作用。     

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5oα. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 (-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION: The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.     

基因重组HBV联合表达反义RNA和显性阴性突变体抗HBV作用及HBV包装细胞系的构建

目的探讨HBV作为基因治疗载体的可能性并检验其联合表达反义RNA和显性阴性突变体抗HBV的作用.方法在表达完整HBV颗粒的质粒上,经基因修饰后联合表达S区反义RNA和核心-p蛋白的融合蛋白,整合于具有HBV复制的2.2.15细胞,形成细胞克隆,ELISA法检测细胞培养上清液中HBsAg和HBeAg,斑点杂交法检测细胞内HBV核壳中HBV DNA,PCR检测上清液中重组HBV颗粒.HBV全基因经删除包装信号ε区后,插入到G418抗性pCI-neo载体,转染HepG2细胞系,用G418筛选形成细胞克隆,检测表达HBsAg及HBcAg较多者作为HBV包装细胞系,进一步转染表达复制缺损型HBV的质粒,经两种抗生素同时筛选,PCR方法观察上清液中的病毒.结果2.2.15-pMEP4组、2.2.15-CP组、2.2.15-SAS组和2.2.15-CPAS组,对HBsAg平均抑制率分别为2.74%±3.83%、40.08%±2.05%(t=35.5,P＜0.01)、66.54%±4.45%(t=42.3,P＜0.01)和73.68%±5.07%(t=51.9,P＜0.01);对HBeAg平均抑制率分别为4.46%±4.25%、52.86%±1.32%(t=36.2,P＜0.01)、26.36%±1.69%(t=22.3,P＜0.01)和59.28%±2.10%(t=39.0,P＜0.01);对HBV复制的抑制率分别为0、82.0%、59.9%和96.6%.在各治疗组培养上清液中均能检测出重组HBV颗粒.证明包装细胞系具有HBsAg和HBcAg表达,pMEP-CPAS质粒转染G418抗性包装细胞系,在细胞培养上清液中检出重组HBV,未检出野生型HBV.结论在同一载体上联合表达S区反义RNA及核心蛋白与部分P蛋白的融合蛋白,具有较单一机制更强的抗HBV作用;经修饰后的HBV基因组在野生型HBV辅助下,仍能包装并分泌完整的HBV样颗粒.包装细胞系能为复制缺损型HBV提供包装,但效率较低.     

肝细胞表面PD-L1的表达及干扰素和HBV对其表达的调节

目的测定肝细胞表面程序性死亡分子受体1（PD-L1）的表达,分析干扰素-α（IFN-α）及乙型肝炎病毒（HBV）对PD-L1蛋白和基因表达水平的调节。方法采用流式细胞术、实时定量PCR等方法,测定PLC/PRF/5细胞表面PD-L1分子的表达;观察不同剂量IFN-α作用下,PLC/PRF/5细胞表面PD-L1表达水平的变化;比较HepG2和HepG2.2.15两种细胞的PD-L1mRNA水平,分析HBV对PD-L1表达水平的调节。结果肝细胞表面组成性地表达PD-L1分子;随着IFN-α作用剂量的增加,肝细胞表面PD-L1的表达水平先升高,随后缓慢降低;转染有HBV基因组的HepG2.2.15细胞,其PD-L1的mRNA水平显著高于不含HBV基因组的同源细胞株HepG2细胞。结论肝细胞表面组成性表达PD-L1分子;干扰素及HBV感染可影响肝细胞PD-L1的蛋白和基因表达水平。     

Elevated expression of bisecting N-acetylglucosaminyltransferase-III gene in a human fetal hepatocyte cell line by hepatitis B virus

BACKGROUND AND AIM: UDP-N-acetylglucosamine: alpha-D-mannoside beta-1,4 N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme in N-glycan biosynthesis. Human GnT-III enzyme activity was found to be elevated in the serum of patients with hepatomas and liver cirrhosis and in hepatocellular carcinoma tissues. Therefore, to understand the relationship between the elevation in GnT-III activity and hepatitis B viral (HBV) hepartocarcinogenesis, we investigated GnT-III gene expression in the HBV-infected cells. METHODS: A cell line, HFH-T1, producing HBV was produced by natural infection of human fetal hepatocytes. A 170-bp band corresponding to the pre-S1 region of HBV was detected in the culture medium by polymerase chain reaction. Virions were also isolated from the culture medium by sucrose density gradient centrifugation. The synthesis of both alpha-fetoprotein and albumin as an indicator that these cells were functional hepatocytes and the extent of differentiation was examined. Polymerase chain reaction and Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III were used for HBV DNA and GnT-III detection. RESULTS: Two types of HBV-related particles were secreted into the culture medium; one was a Dane particle (40 nm in size) containing HBV DNA and the other was a subviral hepatitis B surface antigen particle (20 nm in size) that did not contain the viral genome. The secretion from the cell line was diminished by the number of passages and, thus, this cell was renamed as HFH-T2. A decreased level of the HBV was secreted from the cells after a rest period. HFH-T2 cells showed a weak staining for alpha-fetoprotein and a moderate staining for albumin in the cytoplasm around the nucleus. High levels of a 0.7 kb DNA fragment originating from GnT-III DNA were detected in HFH-T2 cells. Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III showed a single band, corresponding to Mr 63 kDa, whereas aglycosyl GnT-III showed a band at Mr 53 kDa, with a molecular weight difference of about 10 kDa. This indicates that HFH-T2 cells express glycosylated GnT-III. GnT-III activities were 347.2 +/- 53.6 pmol/mg of protein/h in HFH-T2, 276 +/- 26.3 in Hep3B, 252.5 +/- 23.3 in HepG2 and 30.7 +/- 3.4 in NIH-3T3. GnT-III activity was higher in HFH-T2 cells than in the hepatoma cell lines, Hep3B and HepG2. CONCLUSION: A human fetal hepatocyte cell line was transformed by infection with HBV and the cell line expressed high levels of GnT-III as the levels of secretion of HBV decreased. The decrease in HBV secretion from HFH-T2 cells could be due to a high level of expression of GnT-III. Such a cell line could be used to investigate relationships between HBV infection and glycosyltransferase gene expression. Furthermore, this cell line will be useful in future studies on the effect of the expression of GnT-III on other glycosyltransferase.     

Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies

Background: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection.Objective: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs.Methods: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications.Results: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system.Conclusion: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.     

QSG-7701和HepG2细胞支持不同乙型肝炎病毒复制模式及其机制

目的研究QSG-7701及HepG2细胞支持HBV复制模式的差异及其内在机制。方法质粒PUC18-HBV1.2转染QSG-7701与HepG2细胞后定量检测细胞上清液中HBV DNA和HBsAg;采用基因芯片技术比较分析二者基因表达差异并用实时定量PCR验证。结果HepG2细胞在转染后6 d内培养上清液可检出HBV DNA及HBsAg,QSG-7701细胞转染后2周内均可检出HBV DNA及HBsAg,且HBV DNA在10 d内保持相对稳定的高水平复制(1×107～3×107拷贝/ml);基因芯片检测结果示QSG-7701细胞中与HBV生活周期相关的因予如HLF、RXRα、IL-6高表达,而HBxIP、SPIK1为低表达,MMP3不表达。结论QSG-7701支持高水平的HBV复制,并可维持cccDNA池的相对稳定,基因差异表达可能为二者支持不同HBV复制模式提供解释。     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus

Studies of hepatitis B virus (HBV) mutants have been hampered by the lack of a small animal model with long-term infection of cloned HBV. Using a mouse model in which liver cells were highly replaced with human hepatocytes that survived over a long time with mature human hepatocyte function, we performed transmission experiments of HBV. Human serum containing HBV and the virus produced in HepG2 cell lines that transiently or stably transfected with 1.4 genome length HBV DNA were inoculated. Genetically modified e-antigen-negative mutant strain also was produced and inoculated into the mouse model. A high-level (approximately 10(10) copies/mL) viremia was observed in mice inoculated with HBV-positive human serum samples. The level of viremia tended to be high in mice with a continuously high human hepatocyte replacement index. High levels and long-lasting viremia also were observed in mice injected with the in vitro generated HBV. The viremia continued up to 22 weeks until death or killing. Passage experiments showed that the serum of these mice contained infectious HBV. Genetically engineered hepatitis B e antigen-negative mutant clone also was shown to be infectious. Lamivudine effectively reduced the level of viremia in these infected mice. In conclusion, this mouse model of HBV infection is a useful tool for the study of HBV virology and evaluation of anti-HBV drugs. Our results indicate that HBeAg is dispensable for active viral production and transmission.     

慢病毒介导HBV X基因稳定表达HepG2细胞系的建立

目的:构建乙型肝炎病毒X基因(HBV X)重组慢病毒表达载体,建立稳定表达HBVX蛋白(HBx)的HepG2细胞系.方法:应用PCR法从质粒pIERES2-EGFP-HBV中扩增X基因片段,克隆至慢病毒载体pZac2.1,应用PCR、酶切和测序鉴定正确后,经病毒包装,感染HepG2细胞,经嘌呤霉素筛选稳定表达细胞株,RT-PCR、免疫组织化学、Western blot鉴定HBx的表达.结果:酶切鉴定和基因序列测定证实长度为489bp的HBx基因成功克隆至慢病毒表达载体pZac2.1-HBx;重组慢病毒经包装、纯化后获得滴度为1×108TU/mL,通过感染HepG2细胞株和嘌呤霉素筛选,8-10d形成生长形态良好的单克隆细胞株HepG2-HBx;RT-PCR鉴定显示细胞株HepG2-HBx在3d,14d,30d和2mo后均可见稳定表达的HBx mRNA;利用免疫组织化学和蛋白免疫印迹法鉴定,细胞株HepG2-HBx可稳定表达HBx蛋白.结论:成功构建了HBV X重组慢病毒载体,获得稳定表达HBx的HepG2细胞系,为进一步研究HBx的生物学功能及致病机制提供细胞模型.