钙离子、镁离子对戊型肝炎病毒感染PLC/PRF/5细胞的影响

为检测钙离子、镁离子对戊型肝炎病毒(hepatitis E virus,HEV)感染PLC/PRF/5细胞(人肝癌亚历山大细胞)的影响,本研究在各实验组PLC/PRF/5细胞的培养体系中加入等量HEV毒种进行孵育,利用实时荧光定量反转录聚合酶链反应以及酶联免疫法,监测HEV核酸和抗原含量;在HEV感染细胞实验组的维持培养液中分别加入钙离子、镁离子、乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA)和乙二醇二乙醚二胺四乙酸(ethylene glycol tetraacetic acid, EGTA),观察钙离子、镁离子、EDTA和EGTA分别存在以及钙离子、镁离子同时存在的情况下,HEV感染后不同时间PLC/PRF/5细胞内及培养上清液中HEV含量。结果显示,HEV接种细胞后1～24 h,钙离子、镁离子的加入能够促进病毒与细胞的结合,而金属离子鏊合剂的加入抑制了病毒与细胞的结合。HEV感染后2～5周,钙离子、镁离子均能增加PLC/PRF/5细胞培养上清液中产生的病毒,其中钙离子的促进作用更加显著。本研究结果表明钙离子、镁离子能够促进HEV感染细胞,在HEV接种细胞后的培养过程中,添加钙离子、镁离子有助于病毒产生。     

戊型肝炎病毒与UBR2相互作用的初步研究

戊型肝炎病毒(hepatitis E virus, HEV)是一种经肠道传播、严重危害人类健康的病毒性肝炎病原体。HEV不仅可以在肝脏中复制,还可以在肝外组织复制并导致组织损伤,如HEV可在男性生殖系统中复制并导致睾丸损伤,但其发病机制尚不明确。UBR2在生殖系统高表达,是男性减数分裂和精子形成的关键,但其与HEV的相互作用尚不清楚。本文以探究HEV与UBR2之间的相互作用关系为目的,通过构建真核表达载体、转染细胞,利用Western blot技术验证UBR2能否成功过表达。同时,在细胞中加入HEV病毒,并利用Western blot技术探究HEV对UBR2的影响。另外,在HEV感染的新西兰大白兔睾丸组织中,利用免疫荧光共定位技术观察HEV与UBR2的相互作用关系。上述实验成功构建了pEGFP-UBR2质粒,并在A549细胞和Huh 7.5.1细胞中过表达,发现在A549细胞系中,HEV对UBR2有抑制作用;在感染HEV的睾丸组织中发现高表达的UBR2与HEV ORF2衣壳蛋白能够共定位,提示了两者可能存在相互作用。这为深入研究HEV与UBR2的相互作用,以及HEV导致生殖系统损伤的致...     

人肝细胞内戊型肝炎病毒结合蛋白的酵母双杂交筛选

为进一步深入研究戊型肝炎病毒(HEV)感染机制以及致病机理,用酵母双杂交系统从人肝细胞cDNA文库中筛选与戊型肝炎病毒衣壳蛋白E2相互作用的蛋白,核酸序列分析及同源性检索表明,4个克隆与E2相互作用,其中一个克隆与P38IP高度同源,细胞免疫共沉淀实验结果显示:在哺乳动物细胞水平仍能够检测到E2与P38IP片段的特异的相互作用。     

非复制型腺病毒携带miR-1的表达诱导肝癌细胞凋亡

miR-1在多种肝癌细胞中被甲基化沉默,导致其下游多个癌基因MET,FoxP1,HDAC4等大量表达,促进了肝癌的增殖.本研究利用非复制型腺病毒Ad-easy携带miR-1（Ad-miR-1）在肝癌细胞PLC/PRF/5和HepG2中过表达miR-1,探讨miR-1抑制肝癌细胞的作用机制.结果表明,肝癌细胞PLC/PRF/5和HepG2中miR-1表达水平极低,感染Ad-miR-1后miR-1表达水平显著上升.肝癌细胞PLC/PRF/5和HepG2感染Ad-miR-1后,MET和FoxP1的mRNA水平和蛋白表达水平被显著下调,细胞凋亡水平显著增加,肝癌细胞的增殖被抑制. 可见,miR-1在肝癌细胞中可能是一个重要的抑癌因子.同时,利用腺病毒载体携带抗癌的microRNA（如miR-1）,也为今后的基因治疗研究提供了一种新方法.     

长非编码RNAZ38对BT549细胞多能因子表达调控的初步研究

该文旨在研究长非编码RNA Z38对BT549细胞中多能因子表达的影响。通过流式细胞术和实时荧光定量PCR(Real-time quantitative polymerase chain reaction,RT-q PCR)进行Z38与CD44+CD24–/low亚群细胞相关性分析,并利用慢病毒Z38-sh RNA感染BT549细胞以干扰Z38表达,通过RT-qPCR和蛋白质印迹实验检测多能因子表达水平。结果显示,Z38表达水平与CD44+CD24–/low亚群细胞比例存在差异性,以BT549细胞Z38表达水平和CD44+CD24–/low比例相对较高,干扰BT549细胞Z38表达水平后,CD44+CD24–/low比例下降,NANOG和SOX2 m RNA和蛋白质水平皆被下调,而OCT4 m RNA和蛋白质水平无明显变化。干扰Z38影响BT549多能因子的表达,尤其可下调与乳腺癌干细胞分化密切相关的NANOG和SOX2基因表达,提示它可能与乳腺癌干细胞的分化有关。     

CD63/ME491在植入前小鼠胚胎及延迟着床小鼠子宫的表达

目的 研究肿瘤转移抑制因子CD63/ME491 mRNA和蛋白在植入前小鼠胚胎及延迟着床小鼠子宫中的表达规律,探讨其在胚胎着床过程中的作用以及雌激素对其表达的调节.方法 应用RT-PCR、免疫荧光、免疫组化技术观察CD63/ME491 mRNA和蛋白的表达规律.结果 在植入前小鼠胚胎中均有CD63/ME491 mRNA及其蛋白表达.CD63/ME491 mRNA在桑葚胚及囊胚期表达较丰富,CD63/ME491蛋白表达于各期胚胎细胞的胞膜和胞浆;CD63/ME491 mRNA在延迟着床小鼠子宫均有表达,但从D5到D8呈下降趋势,雌二醇(E2)激活后mRNA的表达显著上升(P<0.05).CD63/ME491蛋白在延迟着床D5弱表达于上皮下基质细胞,D6～8表达不明显,E2激活后该蛋白明显表达于上皮下基质细胞的胞膜和胞浆.结论 1. CD63/ME491在植入前小鼠胚胎中呈动态表达,提示它参与了胚胎的发育过程;2. CD63/ME491在小鼠子宫中的表达可能受雌激素调节.     

HIV-1感染对细胞内宿主因子TSG101及与Alix表达的影响

探讨HIV-1感染宿主细胞后对其宿主蛋白肿瘤易感基因101蛋白(Tumor Susceptibility Gene 101,TSG101)及ALG-2相互作用蛋白X(ALG-2-interacting protein X,Alix)表达的影响。以HIV-1感染性克隆病毒pNL4-3感染TZM-bl PM1、Jurkat细胞株和人外周血单个核细胞(PBMCs),感染24h后收获细胞提取总RNA,逆转录PCR检测在RNA水平各因子的表达差异;感染48h后收获细胞提取总蛋白,Western-blot检测各因子在蛋白水平的表达差异。结果显示:HIV-1感染对原代PBMC与细胞系表达Alix与TSG101影响显著不同,细胞系主要表现为下调,而原代PBMC主要表现为TSG101上调;细胞系中的下调又细分为Jurkat细胞的Alix与TSG101的双下调、TZM-bl细胞的Alix单下调以及PM1细胞无影响三种情况。HIV-1感染对细胞宿主分子TSG101及Alix在RNA和蛋白水平的表达均有影响,这种影响因细胞的不同而有差异。HIV-1感染调节Alix与TSG101的机制生物学意义尚有待于进一步阐明。     

SIRT6基因沉默对人肝癌细胞凋亡的影响及其机制研究

该文旨在探讨慢病毒介导的沉默信息调节因子6(silent information regulator 6,SIRT6)基因沉默对人肝癌细胞凋亡的影响及其机制。逆转录PCR(RT-PCR)和Western blot分别检测人肝癌细胞系(SK-Hep-1、Huh-7、PLC/PRF/5、Hep G2)和永生化肝细胞系(MIHA)中SIRT6基因的表达水平;利用慢病毒介导的sh RNA干扰技术靶向沉默SIRT6的表达,并通过RT-PCR和Western blot验证其沉默效率;流式细胞术检测SIRT6基因沉默对人肝癌细胞凋亡的影响,进一步应用RT-PCR和Western blot检测SIRT6基因沉默对凋亡抑制蛋白基因(inhibitor of apoptosis proteins,IAPs)家族m RNA和蛋白质水平的影响;最后,应用流式细胞术分析X连锁凋亡抑制蛋白基因(X-linked inhibitor of apoptosis protein gene,XIAP)在SIRT6基因沉默诱导的肝癌细胞凋亡中的作用。结果显示,SIRT6基因在人肝癌细胞系中表达上调;慢病毒介导的sh RNA能抑制人肝癌细胞中SIRT6基因的表达;沉默SIRT6基因的表达能诱导人肝癌细胞凋亡,并降低XIAP的m RNA和蛋白质水平;过表达XIAP能逆转SIRT6基因沉默所诱导的人肝癌细胞凋亡。该研究结果提示,SIRT6基因沉默可能通过调节XIAP的表达从而诱导人肝癌细胞凋亡。     

携带EGFP基因的戊型肝炎病毒重组质粒的构建及其感染性的验证

为了构建携带增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的戊型肝炎病毒(Hepatitis E virus,HEV)重组质粒,转染人肺癌细胞A549细胞验证其感染性,PCR法分两段扩增HEV全基因组序列和EGFP基因序列,将EGFP报告基因插入到HEV ORF2基因下游,并克隆到体外转录表达载体pGEM-7Zf(+)上。然后利用脂质体转染法将重组质粒转入A549细胞,24h后在荧光显微镜下观察EGFP的表达;转染72h后,利用免疫荧光法检测HEV ORF2蛋白的表达。转染7d后将发生病变的A549细胞收集作为接种物,接种A549细胞验证携带EGFP的HEV-EGFP重组病毒的感染性。结果显示经酶切和测序鉴定携带EGFP的HEV重组表达质粒pGEM-HEV-EGFP构建成功;EGFP基因与HEV在A549细胞中可融合表达;携带EGFP的HEV重组表达质粒转染A549细胞7d后出现病变,并且连续传代3代仍具有感染性。本研究成功构建了携带EGFP的HEV全基因组重组质粒pGEM-HEV-EGFP,并成功感染A549细胞,为进一步研究HEV的复制机制及致病机理奠定基础。     

RNA干扰CD151基因表达抑制肝癌细胞侵袭

目的研究RNA干扰(RNA interference RNAi)抑制CD151表达对人类肝癌细胞迁移侵袭的影响及分子机制。方法将CD151-siRNA在脂质体介导下瞬时转入人肝癌HepG2细胞,倒置荧光显微镜观察转染效率,用qPCR,western blot检测HepG2细胞CD151mRNA和蛋白表达,体外研究肿瘤细胞迁移和侵袭能力,并检测相关信号通路的变化。结果成功转染CD151-siRNA后,HepG2细胞CD151基因的表达与正常对照组和阴性对照组相比,mRNA和蛋白表达水平明显降低(P0.05),细胞迁移和侵袭能力明显下降(P0.05),同时,沉默CD151的表达,FAK,ERK的磷酸化受抑制。结论CD151-siRNA能有效抑制人肝癌细胞CD151基因mRNA和蛋白的表达,通过抑制FAK,ERK蛋白的磷酸化水平,降低细胞的迁移和侵袭力。     

Establishment of hepatitis E virus infection‐permissive and ‐non‐permissive human hepatoma PLC/PRF/5 subclones

PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single‐cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus‐like particles bound non‐permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle.     

CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.     

Permissivity of Primary Human Hepatocytes and Different Hepatoma Cell Lines to Cell Culture Adapted Hepatitis C Virus

Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells’ permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.     

Functional absence of FADD in PLC/PRF/5 hepatoma cells: possible involvement in the transformation to hepatoma in HBV-infected hepatocytes.

The death receptor Fas transduces apoptotic death signaling upon stimulation by Fas ligand and plays a key role in viral hepatitis. When hepatitis-B virus (HBV) infects hepatocytes, the Fas ligand/Fas system responds as the triggering machinery of hepatitis. However, some HBV-infected cells may circumvent Fas-mediated apoptosis and transform to hepatoma cells, as do PLC/PRF/5 hepatoma cells. Therefore, in the present study, we used PLC/PRF/5 hepatoma cells to investigate this ability to avoid Fas-mediated apoptosis. When the cells were treated with an agonistic Fas antibody, they showed resistance to Fas-mediated apoptosis. In contrast, HepG2 cells of the same hepatoma line succumbed. Caspase 3 and 8, which are essential regulators for Fas-mediated cell death, were expressed in both hepatoma cell lines, but only HepG2 cells showed activation of the caspases. A comparison study of expression of other death-associated factors between PLC/PRF/5 and HepG2 cells revealed no apparent differences. However, Far-Western blotting analysis using the Fas death domain (FDD) showed a significant difference. Molecular weight comparison and immunoblotting analysis revealed that PLC/PRF/5 cells lack the FDD-associated protein FADD. In addition, FDD-injected HepG2 cells showed a resistance to Fas-mediated apoptosis, and PLC/PRF/5 cells acquired Fas-sensitivity by FADD injection. Here, we propose that a functional absence of FADD is one of the pathways for the carcinogenesis of HBV-infected hepatocytes.     

Differential Expression of Candidate Virus Receptors in Human T Lymphocytes Prone or Resistant to Infection with Patient-Derived Hepatitis C Virus

Accumulated evidence implies that hepatitis C virus (HCV) infects not only the liver but also the immune system. A lymphocyte-specific CD5 molecule was recently identified as essential for infection of T cells with native, patient-derived HCV. To assess whether the proposed hepatocyte receptors may also contribute to HCV lymphotropism, expression of scavenger receptor-class B type 1 (SR-B1), claudin-1 (CLDN-1), claudin-6 (CLDN-6), occludin (OCLN), CD5 and CD81 was examined by real-time RT-PCR and the respective proteins quantified by immunoblotting in HCV-prone and resistant T cell lines, peripheral blood mononuclear cells (PBMC), primary T cells and their subsets, and compared to hepatoma Huh7.5 and HepG2 cells. SR-B1 protein was found in T and hepatoma cell lines but not in PBMC or primary T lymphocytes, CLDN-1 in HCV-resistant PM1 T cell line and hepatoma cells only, while CLDN-6 equally in the cells investigated. OCLN protein occurred in HCV-susceptible Molt4 and Jurkat T cells and its traces in primary T cells, but not in PBMC. CD5 was displayed by HCV-prone T cell lines, primary T cells and PBMC, but not by non-susceptible T and hepatoma cell lines, while CD81 in all cell types except HepG2. Knocking-down OCLN in virus-prone T cell line inhibited HCV infection, while de novo infection downregulated OCLN and CD81, and upregulated CD5 without modifying SR-B1 expression. Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells. This study narrowed the range of factors potentially utilized by HCV to infect T lymphocytes amongst those uncovered using laboratory HCV and Huh7.5 cells. Together with the demonstrated role for CD5 in HCV lymphotropism, the findings indicate that virus utilizes different molecules to enter hepatocytes and lymphocytes.     

Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme

Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.     

Lipid composition of hepatitis B virus surface antigen particles and the particle-producing human hepatoma cell lines

More than 90% of lipids of hepatitis B virus surface antigen (HBsAg) particles produced by two human hepatoma cell lines (huGK-14 and PLC/PRF/5) were composed of phospholipids, with phosphatidylcholine being the dominant component, accounting for more than 80% of total membrane lipids. Analysis of subclass compositions of phospholipids of HBsAg particles and the host cell lines revealed that 1,2-diacyl glycerophosphocholine was preferentially incorporated into the membrane of the HBsAg particles, although both host cell lines contained extremely high concentrations (more than 60% of total phospholipids) of ether-linked phospholipids. Phospholipids of other hepatoma cell lines (HuH-7, Hep-G2, and huL-1) which were not associated with hepatitis B virus (HBV) infection, were composed mostly of 1,2-diacylglycerophospholipids. Activities of dihydroxyacetone-phosphate acyltransferase, which is known to be an obligatory enzyme in ether lipid biosynthesis, were found to be elevated by three- to fourfold in both huGK-14 and PLC/PRF/5 cells compared to those of other hepatoma cell lines. The results suggest a possible relationship between HBV-induced hepatocellular carcinogenesis and the drastic change in the metabolism of membrane phospholipids.