A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host.
A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host.
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DOI：
10.1186/s12985-016-0519-1
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发表时间：
2016-04-05
期刊：
影响因子：
4.8
通讯作者：
Hongeng S
中科院分区：
文献类型：
--
作者：
Sa-Ngiamsuntorn K;Wongkajornsilp A;Phanthong P;Borwornpinyo S;Kitiyanant N;Chantratita W;Hongeng S
丙型肝炎病毒（Hepatitis C virus，HCV）可引起人类慢性肝病和肝细胞癌.原代人肝细胞作为病毒宿主的使用受到组织供应稀缺的限制。已使用Huh 7来源的肝细胞建立了限制于HCV基因型2a（JFH-1）的培养模型。其他基因型包括野生型病毒不能在Huh 7、Huh7.5和Huh7.5.1细胞中繁殖。功能性肝细胞样细胞（HLC）是从正常人iPS细胞发育而来的，作为HCV感染的宿主。通过选择性肝细胞标志物、CYP 450、HCV相关受体和HCV必需宿主因子鉴定成熟HLC。用JFH-1 HCV RNA转染HLCs或用来自患者血清的HCV颗粒感染HLCs。研究了α-生育酚对HCV感染的增强作用和干扰素-α（INF-α）、利巴韦林、索非布韦对HCV感染的抑制作用。检测HCV病毒载量和HCVRNA水平，评价感染效率。完全发育的HLC表达与原代人肝细胞相当的I、II和III相药物代谢酶、HCV相关受体（claudin-1、occludin、CD 81、ApoE、ApoB、LDL-R）和HCV必需宿主因子（miR-122和SEC 14 L2）。α-生育酚转运蛋白SEC 14 L2在HLCs中表达，但在Huh 7细胞中不表达，与HCVser的有效感染有关。HLCs不仅允许HCV RNA的复制，而且允许HCV颗粒（HCVRNA）的产生释放到培养基中。与经典宿主Huh 7细胞相比，HLC驱动源自JFH-1的HCV 6更高的增殖。JFH-1和野生型HCV感染的HLCs表达HCV核心抗原、NS 5A、NS 5 B、NS 3和HCV阴性RNA。HLC允许来自JFH-1、HCV 3或野生型HCV（基因型1a、1b、3a、3b、6 f和6 n）的整个HCV生命周期。通过将细胞与α-生育酚一起孵育来进一步增加HLC中HCVser的感染。感染细胞的上清可感染HLC和Huh 7细胞。用INF-α和利巴韦林处理感染的HLC，细胞组分和培养基中的HCV RNA均降低。HLCs通过上调TNF-α、IL-28 B和IL-29对HCV或野生型HCV感染作出反应。这种以人肝细胞为宿主细胞的血清源性HCV细胞培养模型为研究HCV的生命周期、HCV相关肝细胞癌的发生和抗HCV新药的筛选提供了一个非常好的系统。本文的在线版本（doi：10.1186/s12985-016-0519-1）包含补充材料，可供授权用户使用。
Hepatitis C virus (HCV) could induce chronic liver diseases and hepatocellular carcinoma in human. The use of primary human hepatocyte as a viral host is restrained with the scarcity of tissue supply. A culture model restricted to HCV genotype 2a (JFH-1) has been established using Huh7-derived hepatocyte. Other genotypes including the wild-type virus could not propagate in Huh7, Huh7.5 and Huh7.5.1 cells. Functional hepatocyte-like cells (HLCs) were developed from normal human iPS cells as a host for HCV infection. Mature HLCs were identified for selective hepatocyte markers, CYP450s, HCV associated receptors and HCV essential host factors. HLCs were either transfected with JFH-1 HCV RNA or infected with HCV particles derived from patient serum. The enhancing effect of α-tocopherol and the inhibitory effects of INF-α, ribavirin and sofosbuvir to HCV infection were studied. The HCV viral load and HCV RNA were assayed for the infection efficiency. The fully-developed HLCs expressed phase I, II, and III drug-metabolizing enzymes, HCV associated receptors (claudin-1, occludin, CD81, ApoE, ApoB, LDL-R) and HCV essential host factors (miR-122 and SEC14L2) comparable to the primary human hepatocyte. SEC14L2, an α-tocopherol transfer protein, was expressed in HLCs, but not in Huh7 cell, had been implicated in effective HCVser infection. The HLCs permitted not only the replication of HCV RNA, but also the production of HCV particles (HCVcc) released to the culture media. HLCs drove higher propagation of HCVcc derived from JFH-1 than did the classical host Huh7 cells. HLCs infected with either JFH-1 or wild-type HCV expressed HCV core antigen, NS5A, NS5B, NS3 and HCV negative-stand RNA. HLCs allowed entire HCV life cycle derived from either JFH-1, HCVcc or wild-type HCV (genotype 1a, 1b, 3a, 3b, 6f and 6n). Further increasing the HCVser infection in HLCs was achieved by incubating cell with α-tocopherol. The supernatant from infected HLCs could infect both naïve HLC and Huh7 cell. Treating infected HLC with INF-α and ribavirin decreased HCV RNA in both the cellular fraction and the culture medium. The HLCs reacted to HCVcc or wild-type HCV infection by upregulating TNF-α, IL-28B and IL-29. This robust cell culture model for serum-derived HCV using HLCs as host cells provides a remarkable system for investigating HCV life cycle, HCV-associated hepatocellular carcinoma development and the screening for new anti HCV drugs. The online version of this article (doi:10.1186/s12985-016-0519-1) contains supplementary material, which is available to authorized users.