Abstract:

Objective To explore the effects of dendritic cells（DCs）from hepatitis B virus（HBV）transgenic mice-stimulated autologous lymphocytes on HBV replication in vitro. Methods DCs from HBVtransgenic mice were induced to maturity by incubation with hepatitis B surface antigen（HBsAg）and hepatitis B core antigen （HBcAg）in vitro. Mature DCs and autologous lymphocytes were co-stimulated to form specific sensitized immune effector cells（IEC），then co-cultured with the human hepatoma cell line HepG2.2.15. Changes in morphology and activity of hepatocytes were then observed，and liver enzyme as well asinflammatory cytokine levels in the culture supernatant were detected using enzyme linked immunosorbentassay （ELISA）. Intracellular HBV DNA and covalently closed circular DNA （cccDNA）concentrationwere measured by real-time PCR. Results Co-stimulation by mature DCs and IEC showed no impact on the morphology and liver enzyme expression level of HepG 2.2.15 cells，but the supernatant HBVDNA and intracellular HBV DNA and cccDNA levels decreased significantly compared with those cells co-cultured with immature DCs. Secretion of inflammatory cytokines in the supernatant showed that when HBV DNAwas highly expressed，the concentration of IFN-γ and IL-2 decreased，while IL-10 was increased. Contrastingly， when HBV DNA had low expression，the concentration of IFN-γ and IL-2 increased，however，the expression of IL-10 decreased. Conclusion Co-stimulation of HBV-related antigen-induced mature DCs and autologous lymphocytes showed inhibitory effects on ex vivo HBV replication，and cytokines were suggested to mediate this effect.

摘要：

目的 探讨转乙型肝炎病毒（HBV）基因的小鼠树突状细胞（DCs）刺激自体淋巴细胞在体外对 HBV 复制的影响。方法 采用转 HBV 基因的小鼠 DCs，在体外用 HBV 表面抗原（HBsAg）和核心抗原（HBcAg）诱导 DCs 成熟。将成熟 DCs 与小鼠自体来源的淋巴细胞共刺激后形成特异性的免疫效应细胞（IEC），再与人肝癌细胞系 HepG 2.2.15 细胞共培养，采用生物化学、流式细胞仪、荧光实时定量聚合酶链反应（RT-PCR）和酶联免疫吸附试验（ELISA）等方法检测肝功能、HBV DNA、共价闭合环状 DNA（cccDNA）水平和炎症相关因子的变化。结果 诱导成熟的 DCs 与 IEC 共同作用 HepG 2.2.15 后，其细胞的形态、酶 学无明显变化，但上清液中 HBV DNA 分泌明显减少，以及细胞内 HBV DNA 和 cccDNA 水平也明显降低，与未成熟 DCs 组比较差异有统计学意义。细胞上清液中炎症相关因子的分泌有明显变化 ：HBV DNA 高表达 时，IFN-γ 和 IL-2 水平下降，而 IL-10 升高；HBV DNA 低表达时，干扰素 -γ（IFN-γ）和细胞白介素 -2 （IL-2）水平上升，而 IL-10 水平下降。结论 HBV 相关抗原刺激成熟的转 HBV 小鼠的 DCs 与自体淋巴细 胞共刺激后形成的特异性 IEC，在体外对 HBV 复制具有抑制作用，细胞因子参与其变化。