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好文推荐 | 一种分化良好、稳定表达hNTCP并支持乙肝病毒高效感染的人肝癌细胞系Huh7D(hNTCP)
at :2017/10/13 10:16:22    

 

原文链接:http://www.virosin.org/html/32/04/10.1007s12250-017-3983-x.htm
 

研究背景

乙肝病毒(HBV)感染可引发广泛的临床表现,包括无症状携带状态到急性或慢性肝炎,以及肝硬化和肝癌。目前,可用的治疗药物如干扰素和核苷类似物(NA)并不能有效地清除病毒。因此,更深入地了解HBV的生命周期有利于制定更有效的治疗策略。

 

为研究HBV的完整生命周期,特别是病毒感染的早期事件,建立一种支持HBV稳定感染的细胞模型显得至关重要。目前,人原代肝细胞(PHHs)(Lempp et al., 2017; Ni and Urban, 2017; Shimura et al., 2017)、HepaRG细胞(Gripon et al., 2002; Schulze et al., 2012)以及树鼩原代肝细胞(PTHs)(Kock et al., 2001; Ren and Nassal, 2001)是研究HBV感染早期阶段的主要细胞模型。PHHs是HBV的天然宿主,被认为是HBV感染的理想细胞模型。然而,PHH存在不易获得、批次差异和体外培养后对HBV的易感性快速丧失等局限性,限制了PHHs的应用。 HepaRG细胞是一种人肝祖细胞系,保留了分化为肝样细胞的能力,并在二甲基亚砜(DMSO)和氢化可的松诱导后可被HBV感染 (Gripon et al., 2002)。然而,由于感染效率低下、诱导过程耗时以及专利保护等原因,限制了HepaRG细胞的广泛应用。树鼩肝细胞(PTHs)是PHHs的替代品,具有容易获得和细胞均一的优点。然而,在PTHs感染过程中加入DMSO和聚乙二醇8000(PEG8000)并不能显著增强HBV的感染效果 (Weizsäcker and Roggendorf, 2005)。此外,人血清可与HBV颗粒竞争结合到PTHs细胞膜上,这与HBV感染PHHs情况不一致,导致实验可信度降低。因此,开发新型的HBV感染模型迫在眉睫


2012年,李文辉等确定了HBV/HDV感染的功能性受体:Na+/牛磺胆酸转运多肽(NTCP)(Yan et al., 2012)。同时,德国Urban研究组也证实了人NTCP(human NTCP, hNTCP)在HBV/HDV入侵中起决定性作用(Ni et al,2014)。在此基础上,这两个课题组相继建立了稳定表达hNTCP(HepG2hNTCP)的HepG2细胞系,该细胞系可支持HBV感染(Yan et al.,2012; Ni et al.,2014)。

 

自然状态下,HBV感染高度分化的人肝细胞,并在其中高效复制,因此高分化的肝细胞相比低分化的肝癌细胞可能更容易建立HBV高效感染与复制(Sainz and Chisari, 2006; Tian et al., 2016)。 DMSO可诱导细胞分化、增强HBV转录与复制,因此通常将其加入到HBV感染的PHHs和HepaRG细胞的培养基中(Gripon et al., 1988; Gripon et al., 2002; Schulze-Bergkamen et al., 2003 )。然而,不同的细胞对DMSO耐受性差异很大。综上所述,对DMSO的耐受程度和细胞的分化状态被认为是建立高效可行的HBV感染模型的限制因素。此外, 对4%PEG8000的耐受程度以及hNTCP表达水平也是影响HBV有效感染的限制因素(Gripon et al., 1993; Yan et al., 2012)。


Huh7是一种肝癌细胞系,最初是1982年从患有肝癌的57岁日本男性体内获得(Nakabayashi et al., 1982),然而不同实验室保存的Huh7细胞存在一定差异。在本研究中,周明等使用不同实验室来源的Huh7肝癌细胞株在2.5% DMSO/4%PEG条件下进行测试,从而获得了一株具有抵抗力、可诱导极化的细胞株(命名为Huh7D),并进一步利用重组慢病毒系统构建稳定表达hNTCP的细胞株(Huh7DhNTCP)。随后,研究团队对Huh7DhNTCP细胞在有无DMSO处理的条件下细胞形态特征和分化相关标记进行了鉴定,并对Huh7DhNTCP细胞的HBV易感性进行评估。研究结果表明,Huh7D细胞可耐受2.5%DMSO/4%PEG8000,而其它细胞则无此耐受性。本研究建立的Huh7DhNTCP细胞稳定表达高水平hNTCP,并在DMSO诱导下迅速转化为一种非分裂、分化良好的细胞表型。最重要的是Huh7DhNTCP细胞支持HBV感染的整个生命周期图一。这种细胞培养系统将有助于分析宿主-病毒的相互作用,并有助于筛选抗病毒药物。

 

图一 Huh7DhNTCP细胞系支持HBV感染的整个生命周期。

 

本文第一作者周明,2015年博士毕业于中国科学院武汉病毒研究所,中山大学-深圳市第二人民医院联合博士后,主持国家自然科学基金青年基金(NTCP参与HBV入侵后生活周期的分子机制,批准号81601760)、中国博士后基金(NTCP调控HBV组装与分泌的分子机制研究,批准号2016M602587),主要研究方向乙肝病毒感染模型、感染限制因子鉴定。

 

通讯作者吴春晨博士,现任中科院武汉病毒研究所副研究员。主要研究方向为肝炎病毒感染与免疫、乙型肝炎病毒变异及耐药机理。目前主持国家自然科学基金、国家重大科技专项子课题等;作为研究骨干参与973计划、国家自然科学基金重点项目等。已发表SCI论文23篇。


摘要:Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus,including the early steps of viral entry.Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG),hNTCP expression,and a differentiated state are the limiting factors for successful HBV infection models.In the present study,we used a hepatoma cell line (Huh7DhNTCP) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection.To achieve this goal,different hepatoma cell lines were tested with 2.5% DMSO/4% PEG8000,and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7DhNTCP) using a recombinant lentivirus system.Then,the morphological characteristics and differentiation molecular markers of Huh7DhNTCP cells with or without DMSO treatment were characterized.Finally,the susceptibility of Huh7DhNTCP cells to HBV infection was assessed.Our results showed that Huh7D cells were resistant to 2.5% DMSO/4% PEG8000,whereas the others were not.Huh7DhNTCP cells were established to express a high level of hNTCP compared to liver extracts,and Huh7DhNTCP cells rapidly transformed into a non-dividing,well-differentiated polarized phenotype under DMSO treatment.Huh7DhNTCP cells fully supported the entire lifecycle of HBV infection.This cell culture system will be useful for the analysis of host-virus interactions,which should facilitate the discovery of antiviral drugs and vaccines.

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