Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Infectious Disease, Anhui Provincial Hospital, Anhui Medical University, Hefei, China.
Virol J. 2017 Oct 25;14(1):205. doi: 10.1186/s12985-017-0874-6.
Hydrodynamic injection (HI) of hepatitis B virus (HBV) mouse model is an useful tool for HBV related research in vivo. However, only 40% of C57/BL6 mice injected with 10 μg HBV genome contained plasmid (pAAV-HBV1.2), serum HBsAg more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks in the previous study.
In this study, C57/BL6 and BALB/c mice were hydrodynamic injected with different doses of pAAV-HBV1.2 plasmid DNA. HBV related serum markers were detected by ELISA. ALT levels in the serum were measured using full automated biochemistry analyzer. HBcAg positive cells in the liver were detected by immunohistochemical staining. The mRNA levels of IRF3, ISGs including ISG15, OAS, PKR and immune factors including IFNγ, TNFα, TGFβ, IL-6, IL-10, PDL1 in liver of the mice were quantified by qRT-PCR.
The results showed that the mice injected with 100 μg high-concentration or 1 μg low-concentration of pAAV-HBV1.2 plasmid DNA did not excert dominant influence on HBV persistence. In contrast, injection of 5 μg intermediate-dose of pAAV-HBV1.2 plasmid DNA led to significant prolonged HBsAg expression and HBV persistence in both C57/BL6 (80% of the mice with HBsAg positive more than 6 months) and BALB/c (60% of the mice with HBsAg positive more than 3 months) mice. IFNγ was significant up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Moreover, PDL1 was significant up-regulated in liver of the mice injected with 5 μg pAAV-HBV1.2 plasmid DNA.
In this paper we demonstrated that, in the HBV HI mouse model, the concentration of injected pAAV-HBV1.2 plasmid DNA contributes to the diverse kinetics of HBsAg and HBeAg in the serum as well as HBcAg expression level in the liver, which then determined the HBV persisternce, while the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence.
乙肝病毒（HBV）小鼠模型的流体动力学注射（HI）是体内研究 HBV 的有用工具。然而，在前一项研究中，只有 40%注射 10μg HBV 基因组的 C57/BL6 小鼠和血清 HBsAg 持续 6 个月以上，而没有 10μg pAAV-HBV1.2 质粒 DNA 注射的 BALB/c 小鼠在 4 周以上血清 HBsAg 阳性。
本研究中，C57/BL6 和 BALB/c 小鼠分别用不同剂量的 pAAV-HBV1.2 质粒 DNA 进行流体动力学注射。通过 ELISA 检测 HBV 相关血清标志物。使用全自动生化分析仪检测血清中 ALT 水平。用免疫组化染色法检测肝内 HBcAg 阳性细胞。用 qRT-PCR 定量检测小鼠肝内 IRF3、ISG15、OAS、PKR 等 ISGs 和 IFNγ、TNFα、TGFβ、IL-6、IL-10、PDL1 等免疫因子的 mRNA 水平。
结果表明，注射 100μg 高浓度或 1μg 低浓度 pAAV-HBV1.2 质粒 DNA 的小鼠对 HBV 持续存在没有明显影响。相反，注射 5μg 中剂量 pAAV-HBV1.2 质粒 DNA 导致 C57/BL6（80%的小鼠 HBsAg 阳性持续 6 个月以上）和 BALB/c（60%的小鼠 HBsAg 阳性持续 3 个月以上）小鼠 HBsAg 表达和 HBV 持续时间明显延长。注射 1μg 或 100μg pAAV-HBV1.2 质粒 DNA 的小鼠肝内 IFNγ 显著上调。注射 100μg pAAV-HBV1.2 质粒 DNA 的小鼠肝内 TNFα 显著上调。此外，注射 5μg pAAV-HBV1.2 质粒 DNA 的小鼠肝内 PDL1 显著上调。
本文在 HBV HI 小鼠模型中证明，注射的 pAAV-HBV1.2 质粒 DNA 浓度影响血清中 HBsAg 和 HBeAg 的动力学以及肝内 HBcAg 的表达水平，从而决定 HBV 的持续存在，而抗病毒因子 IFNγ、TNFα 以及免疫负调节因子 PDL1 在 HBV 持续存在中发挥重要作用。
