Shenzhen Xenotransplantation Research and Development Center, Institute of Translational Medicine, Health Science Center, Shenzhen University School of Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518035, P.R. China.
Clinical Laboratory Center, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, Zhejiang 312000, P.R. China.
Mol Med Rep. 2019 Oct;20(4):3820-3828. doi: 10.3892/mmr.2019.10628. Epub 2019 Aug 29.
Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co‑transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single‑copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP‑recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP‑expressing PPHs with pooled HBV‑positive serum and purified particles was determined. The potential use of HBV‑infected hNTCP‑expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post‑entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e‑antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP‑expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV‑positive sera, but were poisoned by raw HBV‑positive sera. The use of HBV‑infected hNTCP‑expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP‑expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.
原代人肝细胞（PHH）是研究乙型肝炎病毒（HBV）感染和抗病毒药物的“金标准”。然而，PHH 的可用性差、批次间差异以及涉及 PHH 的伦理问题限制了其应用。人牛磺胆酸钠共转运蛋白（hNTCP）作为功能性 HBV 受体的发现，使得开发替代模型来补充 PHH 的应用成为可能。本研究基于单拷贝同源基因评估了七种物种的进化距离。基于进化距离和可用性，分离并感染了 hNTCP 重组慢病毒的 PHH 和原代兔肝细胞（PRH），并测试和比较了两种细胞类型对 HBV 感染的敏感性。此外，还确定了表达 hNTCP 的 PPH 用混合 HBV 阳性血清和纯化颗粒感染 HBV 的效率。评估了表达 HBV 的 hNTCP 的 PPH 用于药物筛选的潜在用途。结果表明，与小鼠和大鼠相比，猪和兔在进化树上与人类的差异更小，这表明猪和兔更有可能促进 HBV 进入后生命周期。在 hNTCP 补充和 HBV 感染后，PPH 和 Huh7D 人肝癌细胞，但不是 PRH，表现出乙型肝炎表面抗原和乙型肝炎 e 抗原分泌增加、共价闭合环状 DNA 形成和感染性颗粒分泌增加。表达 hNTCP 的 PPH 易感染从混合 HBV 阳性血清中纯化的 HBV 颗粒，但被原始 HBV 阳性血清毒害。使用 HBV 感染的 hNTCP 表达 PPH 进行病毒进入抑制剂筛选被证明是适用且可重复的。总之，表达 hNTCP 的 PPH 可能是研究 HBV 感染和抗病毒药物的有价值的工具。
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