hNTCP-expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs
hNTCP-expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs
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DOI：
10.3892/mmr.2019.10628
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发表时间：
2019-10-01
影响因子：
3.4
通讯作者：
Mou, Li-Sha
中科院分区：
文献类型：
--
作者：
Zhou, Ming;Qin, Bo;Mou, Li-Sha
原代人肝细胞(PHHs)是研究乙肝病毒感染和抗病毒药物的“金标准”。然而，可获得性差、不同批次之间的差异以及与PHH有关的伦理问题限制了它们的应用。人类牛磺胆酸钠共转运多肽(HNTCP)作为一种功能性的乙肝病毒受体的发现使替代模型的发展成为补充PHHs的使用。在本研究中，基于单拷贝同源基因对7个物种的进化距离进行了评估。根据进化距离和可得性，分离PHHs和原代兔肝细胞(PRHs)，用重组慢病毒感染PHHs和PRHs，检测和比较两种细胞对乙肝病毒感染的敏感性。此外，还测定了用混合的乙肝阳性血清和纯化的颗粒表达hNTCP的PPHs对乙肝病毒的感染效率。评估了乙肝病毒感染的hNTCP表达PPHs在药物筛选中的潜在用途。结果表明，猪和兔比小鼠和大鼠更接近人类，说明猪和兔更有可能促进乙肝病毒进入后的生命周期。PPHs和Huh7D人肝癌细胞在hNTCP互补和乙肝病毒感染后，表现为乙肝表面抗原和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，而PRHs则无。从混合的乙肝病毒阳性血清中提纯的乙肝病毒颗粒对表达hNTCP的PPHs很敏感，但可被原始的乙肝病毒阳性血清毒化。将表达HBVhNTCP的PPHs用于病毒进入抑制物筛选是可行的和可重复性的。综上所述，hNTCP表达的PPhs可能是研究乙肝病毒感染和抗病毒药物的有价值的工具。
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.