Hepatitis B virus infection and replication in a new cell culture system established by fusing HepG2 cells with primary human hepatocytes
Hepatitis B virus infection and replication in a new cell culture system established by fusing HepG2 cells with primary human hepatocytes
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DOI：
10.1016/j.jmii.2014.08.008
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发表时间：
2016-08-01
影响因子：
7.4
通讯作者：
Ma, Li-Xian
中科院分区：
文献类型：
--
作者：
Sai, Lin-Tao;Yao, Yong-Yuan;Ma, Li-Xian
背景资料：B型肝炎病毒（HBV）感染具有严格的种属特异性和组织特异性，因此以往建立的细胞模型均不能在体外再现HBV的自然感染过程。本研究的目的是建立一个新的细胞系，对HBV易感，并能支持HBV的复制。方法：通过融合原代人肝细胞与HepG 2细胞建立杂交细胞系。将杂交细胞与HBV阳性血清孵育12小时。采用荧光定量聚合酶链反应（QF-PCR）检测HBV DNA。用电化学发光法检测HBsAg和HBeAg。间接免疫荧光法检测核心抗原（HBcAg）。结果：建立了一株新的肝癌细胞株HepCHLine-7，并对该细胞株的HBV共价闭合环状DNA（cccDNA）进行了Southern杂交和实时荧光定量PCR检测。从第2天开始观察细胞外HBV DNA，水平范围为9.80（+/- 0.32）x 10（2）拷贝/mL至3.12（+/- 0.03）x 10（4）拷贝/mL。在感染后第2天检测到细胞内HBV DNA，水平范围为7.92（+/- 1.08）x 10（3）拷贝/mL至5.63（+/- 0.11）x 10（5）拷贝/mL。从第4天至第20天检测培养基中的HBsAg。第5天至第20天HBeAg分泌呈阳性。HBcAg在约20%（+/- 0.82%）的杂交细胞中持续显示阳性信号。结论：HepCHLine-7细胞对HBV易感，并支持HBV的复制。因此，它们适合研究HBV的完整生命周期。Copyright（C）2014，Taiwan Society of Microbiology.出版社：Elsevier Taiwan LLC这是CC BY-NC-ND许可证下的开放获取文章（http：//creativecommons.org/licenses/bync-nd/4.0/）。
Background: Hepatitis B virus (HBV) infection is strictly species and tissue specific, therefore none of the cell models established previously can reproduce the natural infection process of HBV in vitro. The aim of this study was to establish a new cell line that is susceptible to HBV and can support the replication of HBV.Methods: A hybrid cell line was established by fusing primary human hepatocytes with HepG2 cells. The hybrid cells were incubated with HBV-positive serum for 12 hours. HBV DNA was detected by quantitative fluorescence polymerase chain reaction (QF-PCR). HBsAg (surface antigen) and HBeAg (ext r acel l u lar f orm o f cor e ant igen) wer e observed by electrochemiluminescence (ECL). HBcAg (core antigen) was detected by the indirect immunofluorescence technique. HBV covalently closed circular DNA (cccDNA) was analyzed by Southern blot hybridization and quantified using real-time PCR.Results: A new cell line was established and named HepCHLine-7. The extracellular HBV DNA was observed from Day 2 and the levels ranged from 9.80 (+/- 0.32) x 10(2) copies/mL to 3.12 (+/- 0.03) x 10(4) copies/mL. Intracellular HBV DNA was detected at Day 2 after infection and the levels ranged from 7.92 (+/- 1.08) x 10(3) copies/mL to 5.63 (+/- 0.11) x 10(5) copies/mL. HBsAg in the culture medium was detected from Day 4 to Day 20. HBeAg secretion was positive from Day 5 to Day 20. HBcAg constantly showed positive signals in approximately 20% (+/- 0.82%) of hybrid cells. Intracellular HBV cccDNA could be detected as early as 2 days postinfection and the highest level was 15.76 (+/- 0.26) copies/cell.Conclusion: HepCHLine-7 cells were susceptible to HBV and supported the replication of HBV. They are therefore suitable for studying the complete life cycle of HBV. Copyright (C) 2014, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).