摘要
背景自1989年丙型肝炎病毒克隆发现以来,丙型肝炎病毒的研究一直受制于研究模型的缺乏,利用黑猩猩动物模型作为研究手段存在诸多方面的限制,小动物模型因丙型肝炎病毒的严格宿主特异性目前不可获得,因此构建一个简单易行的细胞模型系统对丙型肝炎病毒的研究无疑会起到巨大的推动作用。1999年,Lohmann试验小组构建成功的亚基因复制子是丙型肝炎病毒细胞模型研究的一个重要里程碑,该系统可以研究丙型肝炎病毒RNA的复制机制,但对于丙型肝炎病毒与细胞受体之间的关系研究不能提供帮助,随后出现的丙型肝炎病毒假病毒颗粒虽部分促进了宿主受体探索研究,但始终不能完整模拟丙型肝炎病毒的生活周期。2005年底,美国四个实验小组几乎同时报道了可产生大量有感染性丙型肝炎病毒的细胞模型系统,该系统的构建成功得益于一基因型2a的丙型肝炎病毒病毒株的发现——JFH1,根据该病毒株的cDNA序列体外构建的亚基因复制子不需引入顺应性突变即可大量复制。实验人员通过基因克隆技术通过转染该病毒株的RNA或cDNA至易感细胞,得到具有感染性的丙型肝炎病毒颗粒。此细胞模型系统可产生大量的具有感染性的丙型肝炎病毒,对体外研究病毒的组成,生活周期,致病机制及抗病毒药物的筛选提供了一个简易有效的平台。
目的构建可产生有感染性丙型肝炎病毒的细胞模型,在此模型的基础上研究丙型肝炎病毒的理化特性,组成成分及干扰素omega抑制丙型肝炎病毒的作用。
方法将利用基因克隆技术得到的JFH1丙型肝炎病毒全长RNA通过脂质体转染至Huh7.5细胞内,常规传代培养转染细胞,在不同的时间点留取细胞培养上清、细胞总RNA及总蛋白,分别通过核酶保护分析法及蛋白印迹法检测转染后不同时间点转染细胞内丙型肝炎病毒RNA的量及丙型肝炎病毒蛋白的表达水平,同时用留取培养上清再感染Huh7.5细胞,检测转染细胞上清的感染性,证实该方法可产生有感染性丙型肝炎病毒颗粒。在此模型基础上获得大量含感染性丙型肝炎病毒颗粒的上清,经浓缩纯化后,进行蔗糖密度梯度离心,检测体外获得的含丙型肝炎病毒RNA颗粒的颗粒密度,并用蛋白印迹法探讨丙型肝炎病毒颗粒的组成成分。同时利用此模型及丙型肝炎病毒基因型1b的亚基因复制子系统研究干扰素omega的抗病毒作用,用含不同浓度干扰素omega的培基孵育细胞3天后检测细胞内丙型肝炎病毒RNA及蛋白的水平,以干扰素α-2a为对照,检测细胞内丙型肝炎病毒RNA及蛋白的水平,同时检测细胞内磷酸化的STAT1的水平。
结果通过脂质体转染技术可将全长丙型肝炎病毒RNA转染至易感细胞内,经培养传代约2周,转染细胞内可检测到丙型肝炎病毒蛋白的表达,转染细胞上清再感染易感细胞,可致被感染细胞表达丙型肝炎病毒蛋白及RNA,说明转染细胞上清含有有感染性的丙型肝炎病毒颗粒,证实该系统可作为产生有感染性的丙型肝炎病毒的细胞模型。在此细胞模型基础上,我们发现含丙型肝炎病毒RNA的颗粒密度分布广泛,在1.08-1.176g/ml之间,而且低密度颗粒的感染性高于高密度颗粒,在含丙型肝炎病毒RNA的颗粒中可以检测到apoE蛋白的存在,提示apoE为病毒颗粒的组成成分;同时在含丙型肝炎病毒RNA的颗粒中,我们同样可以检测到病毒的NS2,NS3,NS4B,NS5A蛋白,这提示上述蛋白亦可能为病毒的组成成分。最后,利用丙型肝炎病毒基因型1b的亚基因复制子系统及丙型肝炎病毒的细胞培养模型,我们证实omega干扰素同样具有很强的抗病毒作用,两种干扰素处理后细胞内丙型肝炎病毒的RNA和蛋白的水平较空白对照组均明显下降,p＜0.05,且未发现细胞毒性;与相同浓度的干扰素a—2a比较,omega干扰素处理组细胞内蛋白浓度明显较干扰素a—2a处理组为低,其差异具有统计学意义(p＜0.05);而且比较两种干扰素对丙型肝炎病毒RNA抑制作用的EC50,omega干扰素的EC50较a—2a干扰素约低10倍。在丙型肝炎病毒的细胞培养模型基础上,两种干扰素处理感染性模型系统后上清感染性均较对照组减弱,p＜0.05;同样的,omega干扰素处理组的感染性下降得较a—2a干扰素处理组更为明显,p＜0.05。两种干扰素处理后细胞内磷酸化的STAT1的水平均较对照组为高,p＜0.05;而且omega干扰素处理组较a—2a干扰素处理组升高得更为明显,p＜0.05。
结论1.可通过脂质体转染技术成功将约10kb的丙型肝炎病毒(JFH1病毒株)全长RNA转染入Huh7.5细胞。2.JFH1病毒株病毒RNA转染后可在细胞中自我复制、翻译表达丙型肝炎病毒特异性蛋白;同时可获得有感染性的丙型肝炎病毒颗粒的培养上清。3.体外培养获得的含丙型肝炎病毒RNA的颗粒以多种密度形式存在,且以低密度为主。低密度病毒颗粒的感染性高于高密度病毒颗粒。4.apoE存在于某些含丙型肝炎病毒RNA的颗粒中,其含量的多少与丙型肝炎病毒颗粒的感染性高低存在良好的正相关关系。5.除外结构蛋白,部分传统意义上的非结构蛋白包括NS2、NS3、NS4B、NS5A亦可在HCV病毒颗粒中检测到,其生物学意义有待进一步研究。6.干扰素omega具有抑制丙型肝炎病毒基因型1b及2a病毒复制的作用,且与干扰素α-2a比较,其抗病毒活性强于干扰素α-2a,原因可能与其激活干扰素受体的能力更强有关。
Background From 1989 that HCV clone was defined first time,the investigation in the field of HCV was limited by the studying model. Chimpanzee is the only animal model,but because of high expense and other ethical reasons,this huge animal model could not used widely,it was very imperious to construct a celll culture system of HCV.In 1999, Lohmann et al first reported the HCV 1b subgenomic replicon and could uesde for the research of HCV replication,later the HCV pseudopartical came out,it was useful for defining the cellular receptor interacting with HCV membrane proteins.Although the subgenomic replicons and the HCV pseudoparticals were complementary partly,they both could not produce infectious virus,could not imitate the complete whole life cycle of HCV.Till 2005,different four groups reported the HCVcc(HCV celll culture) system that could robustly produce infectious HCV virions,this system benefited from the separation of one viral clone named JFH1(Japanese fulminant hepatitis 1,JFH1),its subgenomic replicon could replicate efficiently without any adaptive mutation.This celll culture system greatly facilitate the investigation of determining the properties and biochemical compositions of HCV virions,studying the whole HCV life cycle and pathogenic mechanism and very importantly,it supplies the platform for antiviral drug selection
Objective To construct the HCV cell culture system and based on HCVcc,exploring the properties and biochemical compositions of HCV virions,and evaluating the antiviral effect of interferon omega.
Mathods Transfected the JFH1 HCV full-length RNA (HCV-FL-RNA) produced by reverse genetics into Huh7.5 cells with DMRIE-C liposomal transfection reagent,passaging the transfected cells normally and harvestd the cellular RNA、cellular protein and cell culture supernatant at different time point。Detected HCV RNA and protein by ribozyme protection assay and western blotting,at the same time,infected the na(i|¨)ve Huh7.5 cells with harvested supematant,confirming that this transfection system could produce infectious HCV virions.Based on this system,got a large quantity of supernatant containing infectious HCV particles,after concentration and purification,tht concentrated HCV was subjected to 20 to 60%sucrose gradient sedimentation analysis,a total of 11 fractions with 1 ml each were collected from the bottom to the top of the sucrose gradient,The sucrose in fractions was removed by dilution with 1x phosphate-buffered saline(PBS) at a 1:12 ratio and then ultracentrifugation at 40,000 rpm for 16 h at 4℃in a Beckman SW41 rotor,the resulting HCV pellets were resuspended and aliquoted for subsequent studies.We detected the density and infectivity of each fraction and detected some HCV nonstructure proteins of combined infectious fractions by western blotting.At the same time,we tested the antiviral effect of interferon omega based on HCV 1b replicon and 2a cell culture system.
Results Through liposomal transfection reagent we could transfected huge size nucleic acid into susceptible cells.About 2 weeks after transfection passaging,we could detect the obvious HCV RNA and proteins in the transfected cells,and the supernatant collected from the transfected cells could infect the naive Huh7.5 cells confirmed by detecting HCV RNA and proteins in the Huh7.5 cells incubated with the supernatant,the result mentioned above showed that this transfection method could produce infectious HCV virions and we could use this HCV cell culture model to harvest a large quantity of supernatant containing infectious HCV particles.Based on this system,we found that HCV viral particles displayed a large range of densities,varied from 1.08 g/ml to 1.176 g/ml,as revealed by density gradient sedimentation analysis, and the HCV infectivity correlated inversely with the density of HCV particles.We detected apoE protein in some fractions and it showed that apoE amout was correlated with the fraction's infectivity.Interestingly, we also detected some nonstructure protein-NS2、NS3、NS4B、NS5A in the combined infectious fractions,this suggested that except for core、membrane protein 1 and 2,some traditional nonstructure proteins and host proteins such as apoE were also a part of HCV virion.At the same time we tested the antiviral effect of interferon omega based on HCV 1b replicon and 2a cell culture system compared with interferonα-2a,at the same concentration,the HCV RNA and protein levels treated with interferon omega were lower than that treated with interferonα-2a,the difference was statiatic significant(p＜0.05),and based on the HCV RNA analysis,EC50 of interferon omega was lower than interferonα-2a.The infectivity of supernatant from interferon treated groups were decreased compared with control(p＜0.05),especially the group treated with interferon omega.Those difference may be due to the higher level of intracellular STAT1 treated with interferon omega.
Conclusions 1.Through liposomal transfection reagent,huge size nucleic acid could be transfected into susceptible cells.2.After HCV-FL-RNA tansfected into Huh7.5 cell lines,the exogenic RNA could replicate automatically and express proteins;the supernatant from transfected cells contains infectious viral particles.3.HCV virions obtained from HCV cell culture system displayed a large range of densities,and the HCV infectivity correlated inversely with the density of HCV particles.4.ApoE was found in some HCV particles and its amount correlated with the infctivity of HCV particles.5,except for structure proteins,some traditional nonstructure proteins including NS2、NS3、NS4B、NS5A coud be detected in HCV virion.6.Interferon omega could suppress the replication of HCV(genotype 1b and 2a),and its antiviral activity was stronger than interferonα-2a,this difference may be due to the stronger stimulation of interferon receptor.
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