转基因小鼠特异性免疫效应淋巴细胞影响乙型肝炎病毒复制的体外研究  

Study on the impact of specific sensitized effector cells on hepatitis B virus replication in vitro

作　　者：宋红丽[1] 郑卫萍[1] 杨洋[1] 刘涛[1] 吴本娟[1] 付楠楠[2] 张友成[1] 沈中阳[1] 

机构地区：[1]天津市第一中心医院器官移植中心,天津市器官移植重点实验室,天津300192 [2]天津市儿童医院,天津300074

出　　处：《实用器官移植电子杂志》2015年第4期200-208,共9页Practical Journal of Organ Transplantation(Electronic Version)

基　　金：国家自然基金面上项目(81270528;81170444);天津市应用基础及前沿技术研究计划重点资助项目(08JCYBJC08400;11JCZDJC27800;12JCZDJC25200);天津市卫生局科技基金攻关项目(05KYZ24;2011KY11;10KG101)

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(HBs Ag)和核心抗原(HBc Ag)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Hep G 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(ccc DNA)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Hep G 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ccc DNA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。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 HBV transgenic mice were induced to maturity by incubation with hepatitis B surface antigen（HBs Ag） and hepatitis B core antigen（HBc Ag） 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 Hep G 2.2.15. Changes in morphology and activity of hepatocytes were then observed,and liver enzyme as well as inflammatory cytokine levels in the culture supernatant were detected using enzyme linked immunosorbent assay（ELISA）. Intracellular HBV DNA and covalently closed circular DNA（ccc DNA） concentration were 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 Hep G 2.2.15 cells,but the supernatant HBV DNA and intracellular HBV DNA and ccc DNA levels decreased significantly compared with those cells co-cultured with immature DCs. Secretion of inflammatory cytokines in the supernatant showed that when HBV DNA was 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.

关 键 词：树突状细胞 乙型肝炎病毒 淋巴细胞 病毒复制 免疫调节 

分 类 号：R373.21[医药卫生—病原生物学]