体外感染实验探讨丙型肝炎病毒的ADE分子机制

目的研究丙型肝炎病毒(HCV)在感染人滋养层细胞的过程中是否存在抗体依赖性感染增强(ADE)作用,以探讨HCV母婴传播的分子机制。方法将HCV阳性血清以4种不同方式感染人滋养层细胞,以免疫电镜观察HCV病毒颗粒在滋养层细胞中的表达,应用RT-PCR法、免疫组化法检测细胞内外的HCVRNA正链、负链,HCVNS5、NS3及C区抗原。结果在滋养层细胞胞浆内发现了HCV病毒颗粒,HCVRNA正链、负链,HCVNS5、NS3、C区抗原仅在全血清感染细胞内或上清中间断测得。结论HCV可以在滋养层细胞中复制。HCV感染滋养层细胞的过程中存在ADE机制,抗体和补体均参与了HCV的跨膜转运过程。     

丙型肝炎患者外周B淋巴细胞系及HCV的形态学研究

研究ＨＣＶ对人Ｂ淋巴细胞的感染 ,建立ＨＣＶ感染的丙型肝炎患者Ｂ淋巴细胞模型 (ＣＢＣＬ) ,并进行ＨＣＶ的形态学研究 ,观察ＨＣＶ在其中的复制及ＨＣＶ形态学及超微结构。用ＥＢ病毒转化Ｂ细胞建立Ｂ细胞系 ,以逆转录聚合酶链反应 (ＲＴ ＰＣＲ)、免疫组织化学、原位杂交方法检测Ｂ细胞系上清液及细胞内的ＨＣＶ抗原及ＨＣＶＲＮＡ ,并通过电镜对ＨＣＶ进行形态学研究。用ＥＢ病毒转化丙型肝炎患者Ｂ淋巴细胞传代培养 ,使其成为永生细胞系。用抗ＨＣＶ ＮＳ3、ＮＳ5、ＣＯＲＥ单克隆抗体检测ＣＢＣＬ中ＨＣＶ抗原 ,发现阳性颗粒主要见于…     

初步探讨丙型肝炎病毒体外感染的致病机理

目的:初步探讨原代培养肝细胞对HCV的易感性,并在此基础上观察HCV感染后肝细胞形态学的变化。方法:用HCV RNA阳性血清与培养肝细胞共同孵育后,收集不同时相点标本,以逆转录聚合酶链反应(RT-PCR)、分别细胞内或上清中正负链RNA,免疫组化检测细胞内HCV NS3、NS5抗原的表达以及原位杂交检测细胞内HCV负链RNA,同时在电镜下观察细胞形态学变化。结果:接种感染血清后第3～5d,即可在细胞内或培养上清中检出HCV正负链RNA;感染肝细胞内可见HCV NS3、NS5抗原表达,阳性物质位于胞浆中;原位杂交法证实细胞内存在负链RNA,也位于胞浆中;光镜下未发现感染细胞形态学变化,电镜下发现胞浆内含有大量的脂肪空泡、多泡体,并可见疑似病毒样结构。结论:HCV不但能感染树鼩肝细胞,而且在体外能直接致细胞病变。     

体外培养人胎盘滋养层细胞内吞丙型肝炎病毒过程的电镜观察

目的 探讨丙型肝炎病毒（HCV）感染体外分离培养人胎盘滋养层细胞的可能机制，观察HCV感染滋养层细胞后在细胞内的具体定位。方法采用胰蛋白酶消化法及Percoll密度梯度分离法分离培养人胎盘组织中滋养层细胞后，以HCVRNA阳性血清对滋养层细胞进行体外感染实验，不同时间终止实验，制备电镜标本，透射电镜观察。结果HCV感染滋养层细胞的过程中有吞噬小泡形成，形态学上符合被覆小凹的特点；感染后HCV病毒样颗粒多定位于粗面内质网附近。结论HCV可以感染滋养层细胞，其感染过程可能是被覆小凹介导的内吞过程。本研究提供了HCV体外感染人胎盘滋养层细胞具体方式的形态学资料，对HCV宫内传播机制的进一步研究奠定了实验基础。     

肝脏疾病的病理学现状

一、丙型肝炎电镜检查感染丙型肝炎病毒 (HCV )的人类和黑猩猩的肝脏发现平行排列的短管结构 (病毒外壳物质 )和直径为 5 0～ 6 0nm的病毒样颗粒。感染HCV的人 ,其淋巴细胞内显示有这种短管的聚集。慢性丙型肝炎肝活检标本常常显示肝脂肪变性 ,且基因表型 3a和体重指数 (BMI)与脂肪变性密切相关。HCV核心蛋白通过导入氧化趋势和活化氧参与脂肪变性。这样依次产生线粒体渗透性改变、细胞色素C释放 ,最终细胞发生凋亡。与转基因小鼠情况相似 ,载脂蛋白正常转运脂质的作用受到HCV蛋白的抑制 ,其中HCV核心蛋白影响载脂蛋白A2 ,非结构抗原 5A(NS5A)则作用于载脂蛋白A1。另有一篇文章进一步讨论了HCV相关脂肪变性的可能机制及HCV核心蛋白与肝细胞肝癌的关系。基因片段分析显示HCV导致的肝硬化与某些基因表达上调有关。对血清转氨酶水平正常的HCV携带者随访 6个月以上 ,约有 2 0 %的携带者出现血清转氨酶升高。流行病学调查显示 ,HCV感染与B淋巴细胞异常增生有关 ,并发现 7例绒毛淋巴细胞型脾淋巴瘤 (脾边缘区淋巴瘤 )患者经α2b干扰素治疗病情彻底缓解 ,其HCVRNA检测为阴性。丙型肝炎患者...     

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。     

人肝癌细胞株7721HCV体外感染模型的建立

目的建立接近体内自然感染状态并能稳定支持HCV体外长期复制的感染细胞模型。方法将慢性丙型肝炎患者的血清与人肝癌细胞系7721共同孵育8小时后,分别以反转录-聚合酶链反应、免疫组化、原位杂交检测细胞和/或培养上清中的HCV正、负RNA、HCV抗原的表达及HCV-RNA在感染细胞内的定位。结果感染血清和细胞共同孵育后的第2～65天,从细胞内和培养上清中均可间断地检测出HCV正、负RNA,即使其间细胞传代4次HCV NS_3抗原、NS_5抗原在细胞内能稳定表达,原位杂交显示HCV RNA阳性物质多位于细胞浆。结论 7721细胞不但对HCV易感,而且可以稳定地支持HCV的体外长期复制,此模型可以用于HCV感染、复制机理的研究、抗病毒药物的评价及保护性抗体/疫苗的初步筛选。     

商陆抗病毒蛋白抗HCV作用的初步研究

目的　探讨商陆抗病毒蛋白 (PAP)对HCV的抑制作用。方法　用不同浓度PAP处理HCV感染细胞模型 ,用荧光定量PCR检测HCVRNA。结果　HepG2 细胞内HCV持续表达达 3 0d以上。PAP处理HCV HepG2 细胞后 ,各组细胞内HCVRNA含量在 48h、96h和 14 4h差异非常显著 (P <0 .0 1)。培养上清液HCVRNA含量在 48h时 ,各组间无明显差异 (P >0 .0 5 ) ,但在 96h和 14 4h时 ,各组间差异非常显著 (P <0 .0 1)。此种抑制作用随PAP浓度的增加而增强。结论　PAP具有抑制HCV复制和表达的作用。     

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自丙型肝炎病毒(hepatitis C virus,HCV)发现以来,关于其病毒学的研究不断取得一些进展,这些进展主要依赖于对其分子生物学的研究以及对其相关基因表达产物的研究。而关于HCV的形态还没有取得一致的可重复性的结论。这些HCV病毒学的进展主要在以下几方面与临床有关。1抗HCV药物筛选模型的建立病毒的复制有赖于感染活的宿主细胞,为了建立有效的抗HCV复制药物的细胞筛选模型,迫切需要建立能够允许HCV持续感染并有效复制的细胞系,但HCV RNA感染或转染原代培养的肝细胞、传代非瘤肝细胞系(PH5CH)、肝癌细胞系(Huh7)、逆转录病毒感染…     

HCV感染人滋养层细胞过程中抗体依赖性增强作用的初步研究

目的研究丙喇肝炎病毒（HCV）在感染人滋养层细胞的过程中是否存在抗体依赖性感染增强（ADE）作用，探讨HCV母婴传播的分子机制。方法将HCV阳性血清以4种不同方式感染体外培养人滋养层细胞，应用RT-PCR法对标本进行HCV RNA定性检测，并进一步评估不同浓度CD16单抗对感染过程的阻断作用。此外，应用免疫电镜技术观察滋养层细胞感染HCV病毒颗粒情。结果HCV RNA在全血清组、CD81单抗阻断组及LDL-R单抗阻断组多个标本均为阳性表达，且平均拷贝数较高，在FcγRⅢ（CD16）单抗阻断组多个标奉内未能检测到HCV RNA的表达，且阳性标本拷贝数较低；CD16单抗对感染的阻断作用随浓度的升高而增强；在全血清组、CD81单抗阻断组及LDL-R单抗阻断组多个标本细胞内可间断测得未脱壳的HCV病毒颗粒，CD16单抗阻断组细胞内未能检出。结论滋养层细胞膜CD81和LDL-R分子不参与HCV感染滋养层细胞的过程，而FcγRⅢ分子则与此过程密切相关。HCV感染人滋养层细胞过程中存在抗体依赖性增强作用。     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

Serum‐derived hepatitis C virus 1a infection of human astrocyte cell line SVG

Neuroinvasion of hepatitis C virus (HCV) is evidenced by recent clinical studies. In this study, serum‐derived HCV infection of astrocytes was analysed. Astrocytes were infected with HCV‐positive serum, and viral replication was assessed on different days postinfection. RT‐PCR was positive for HCV‐negative strand on 5th and 7th day postinfection in the HCV‐positive serum‐infected astrocytes. Real‐time RNA count in the cell culture supernatant was steadily increasing from day 3 to day 7. To reconfirm the viral replication, astrocytes were treated with an antiviral before the serum infection, and the antiviral treatment significantly reduced the viral RNA count. Further, the virus‐infected cells stained positive for the presence of viral core protein. Electron microscopy revealed the presence of HCV‐like particles in the astrocyte cell culture supernatant. In conclusion, serum‐derived HCV replicates in human astrocyte cell line SVG.     

Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients.

Hepatitis C virus (HCV) is the primary agent of posttransfusion non-A, non-B hepatitis. HCV RNA was detected in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction in 17 of 24 HCV-infected patients with chronic hepatitis with or without cirrhosis. One of 5 patients whose PBMC contained HCV RNA also had negative-stranded HCV RNA in the PBMC. In 3 of 11 patients whose PBMC contained HCV RNA, flow cytometry with a murine monoclonal antibody to HCV core epitope revealed cytoplasmic staining of peripheral blood monocytes. The monocyte surface and the peripheral blood lymphocytes did not stain for HCV core epitopes. No correlation could be made between the presence of HCV RNA or antigen in PBMC and any serologic markers of HCV infection. These results indicate that monocyte uptake of HCV by either phagocytosis or infection may be part of the pathophysiology of this chronic disease.     

Occult persistence and lymphotropism of hepatitis C virus infection

Recent discovery of occult hepatitis C virus （HCV） infection persisting after spontaneous or antiviral therapy-induced resolution of hepatitis C was made possible by the introduction of nucleic acid amplification assays capable of detecting HCV RNA at sensitivities superseding those offered by clinical tests. Although individuals with this seemingly silent HCV infection are usually anti-HCV antibody reactive and have normal liver function tests, occult HCV infection has also been reported in anti-HCV-negative individuals with persistently elevated liver enzymes of unknown etiology. Studies have shown that HCV RNA can persist for years in serum, lymphomononuclear cells and liver in the absence of clinical symptoms, although histological evidence of a mild inflammatory liver injury can be occasionally encountered. Furthermore, while HCV RNA can be detected in circulating lymphoid cells in approximately 30% of cases, a short-term culture under stimulatory conditions augments HCV replication in these cells allowing detection of virus in otherwise HCV-negative cases. HCV infects different immune cell subsets, including CD4^＋ and CD8^＋ T lymphocytes, B cells and monocytes. Studies employing clonal sequencing and single-stranded conformational polymorphism analyses have revealed unique HCV variants residing in immune cells, further strengthening the notion of HCV lymphotropism. Overall, the data accumulated suggest that occult HCV infection is a common consequence of resolution of symptomatic hepatitis C and that examination of the cells of the immune system is an effective approach to diagnosis of HCV infection and its long-term persistence. Further work is required to fully realize pathogenic and epidemiological consequences of occult HCV persistence.     

Hepatitis C virus particles and lipoprotein metabolism

The majority of infectious hepatitis C particles are present in the low-density fractions from plasma of infected patients, suggesting an association of the virus with lipoproteins and the use of lipoprotein receptors for cell entry. Although classical hepatitis C virus (HCV) virions have been reported by some investigators, their role in the HCV life cycle has not been clearly identified. Moreover, two other forms of particles have been characterized: low-density lipo-viro-particles (LVPs) and high-density particles. The latter are nonenveloped nucleocapsids that have immunoglobulin G Fcgamma binding capacity. LVPs are spherical particles enriched in triglycerides. At a minimum, they contain apolipoprotein B, HCV RNA, and core protein. The main source of LVPs is likely to be the enterocytes rather than the hepatocytes, suggesting an interaction between chylomicron and LVP assembly. In experimental systems, HCV core protein inhibits the microsomal triglyceride transfer protein, binds to apolipoprotein AII, and induces accumulation of cytoplasmic lipid droplets. A model of LVP and HCV core-lipid droplet generation is proposed.     

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes

HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和