【作者】 郑建铭；

【导师】 施光峰；

【作者基本信息】 复旦大学 ， 内科学， 2010， 硕士

【摘要】 目的：通过观察乙型肝炎病毒干预后小鼠肝脏树突状细胞干扰素调节因子3表达的变化,探讨HBV持续感染分子免疫机制。方法：采用细胞滤网过滤、Percoll密度梯度离心和免疫磁珠分选法分离肝脏树突状细胞,并用粒细胞—巨噬细胞集落刺激因子和白介素4诱导培养。将树突状细胞分为两组：一组与HBV共培养,另一组加入等量的细胞培养液作为正常对照组。24h后,两组均加入Poly I:C刺激,收集不同时间点细胞和上清,留细胞培养液作为空白对照,用western blot检测IRF3表达,用ELISA法检测上清中IFN-β浓度。结果：1.DC与HBV共培养前,IRF3表达弱。用poly I:C刺激DC后,IRF3表达明显增加,呈时相性,在2-6h达到高峰。DC与HBV共培养后,再用poly I:C刺激,IRF3表达的时相性不明显,未见明显升高。DC表达IRF3的能力与病毒载量有关,高病毒载量能明显抑制IRF3表达,病毒载量低于106 copies／ml时差异不明显。2.0h时HBV组培养液上清IFN-β浓度(12.38±3.71pg／ml)与正常对照组(10.83±4.11 pg／ml)相比,差异无统计学意义(t=0.8398,P>0.05)。Poly工：C刺激后6h,HBV组培养液上清IFN-β浓度(88.67±9.01 pg／ml)与正常对照组(137.68±12.28 pg／ml)相比,差异有统计学意义(t=9.653,P<0.01)。Poly I:C刺激后24h,HBV组培养液上清IFN-β浓度(69.89±5.80 pg／ml)与正常对照组(72.25±8.61 pg／ml)相比,差异无统计学意义(t=0.6820,P>0.05)。结论：1.用poly I:C刺激正常DC后,IRF3表达增加,呈时相性。2.HBV干预后,用poly I:C刺激DC后,IRF3表达增加不明显。高载量HBV能明显抑制肝脏树突状细胞IRF3的表达。3.HBV干预后,肝脏树突状细胞IRF3下游IFN-β表达降低。更多还原

【Abstract】 Objectives:To investigate the molecule immunology mechanism of HBV persistent infection, we observed the expression of IRF3 in murine liver dendritic cells, which were intervened by HBV virions.Methods:The murine liver dendritic cells were isolated via anti-CD11c microbeads, and were incubated with GM-CSF and IL-4 to induce the DCs generation and proliferation. DCs were divided into two groups. One group was cultured with HBV virions in 24h; the other group was cultured without HBV as a normal control group. Then, harvesting the cells and supernatants were detected the expressions of p-IRF3 by western blot and the concentration of IFN-βby ELIS A, after stimulated by poly I:C.Results:1. The expression of IRF3 was too weak to be detected by western blot, before dendritic cells were co-incubation with HBV. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C. The highest expression time-point was between 2h to 6h. The expression of IRF3 was not significantly increased after dendritic cells were incubated with HBV. The ability of expression of IRF3 of DCs was correlated with HBV viral load. High HBV viral load can significantly inhibit expression of IRF3. HBV did not inhibit IRF3 expression significantly, if HBV viral load was lower than 106 copies/ml.2. IFN-βconcentration was 12.38±3.71pg/ml at Oh,88.67±9.01 pg/ml at 6h, 69.89±5.80 pg/ml at 24h in supernatants of HBV group, and was 10.83±4.11 pg/ml at 0h,137.68±12.28 pg/ml at 6h,72.25±8.61 pg/ml at 24h in supernatants of normal control group, respectively. There was no statistically significant difference at 0h(t=0.8398, P>0.05) and at 24h(t=0.6820, P>0.05) between two groups. But, there was statistically significant difference at 6h(t=9.653, P<0.01) between two groups. Conclusion1. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C and had a relationship with different stimulated time-point.2. After co-incubation with HBV, the expression of IRF3 in liver DCs was not increased significantly, compared with normal control. High HBV viral load can inhibit the expression of IRF3 significantly.3. The secretions of IFN-β, downstream of IRF3, were decreased in murine liver dendritic cells after intervened by HBV virions.更多还原