乙型肝炎病毒干预后小鼠肝脏树突状细胞P-干扰素调节因子3表达的研究

Study on type I interferon and phospho-IRF3 in murine liver dendritic cells after intervened by HBV

目的研究HBV干预后小鼠肝脏树突状细胞矿干扰素调节因子3及其下游I型干扰素表达的变化。方法用免疫磁珠分选法分离肝脏树突状细胞，并用粒细胞一巨噬细胞集落刺激因子和自细胞介素4在24孔板中诱导培养。用超离心技术获得HBV，并用实时荧光定量PCR检测。第5天将培养的树突状细胞分为2组：HBV组与HBV共同培养，对照组加入等量的细胞培养液。24h后，两组均加入聚肌胞刺激，收集0、l、2、6、24h的细胞和上清液，用Westernblot检测P-千扰素调节因子3的表达情况，用酶联免疫吸附法检测上清液中干扰素β的浓度。组问比较用f检验。结果0h时HBV组培养液上清液干扰素β浓度【（12．38±3．71）pg／ml】与对照组【（10．83±4．11）pg／m1]比较，差异无统计学意义（P〉0．05）。聚肌胞刺激后6h，HBV组培养液上清液干扰素D浓度[（88．57±9．01）pg／ml】与对照组[（137．58±12．28）pg／m1]比较，t=9．653，P〈0．01，差异有统计学意义。聚肌胞刺激后24h，HBV组培养液上清液干扰素p浓度[（69．89±5．80）pg／ml】与对照组【（72．25±8．61）pg／m1]比较，差异无统计学意义（尸〉0．05）。聚肌胞刺激小鼠肝脏树突状细胞后P-干扰素调节因子3表达逐渐增强。经HBV干预在感染时病毒与细胞数量的比值为25，24h后再用聚肌胞刺激2h，HBV组P干扰素调节因子3表达较对照组减弱。结论HBV干预后小鼠肝脏树突状细胞P-干扰素调节因子3及其下游I型干扰素表达均下调。

Objective To detect the secretions of type I interferon and the expressions of phospho- IRF3 in routine liver dendritic cells intervened by HBV. Methods The murine liver dendritic cells were isolated via anti-CD1 lc microbeads and were incubated in the presence of GM-CSF and IL-4 to induce the DC generation and proliferation in 24-well cell culture plates. HBV virions were isolated via ultracentrifuga- tion and were detected by quantitative Realtime-PCR. The DCs were divided into two groups： one group was cultured with HBV virions for 24 hours, the other group was cultured without HBV as control group. The cells were harvested at Oh, lh, 2h, 6h and 24h after being stimulated with poly I：C and the expressions of p- IRF3 and the concentration of IFNβ in supernatants were detected with western blot and ELISA respectively. Results The IFN βconcentrations at 0 h, 6 h and 24 h in the supernatants of the HBV group and the control group were （12.38±3.71） pg/ml, （88.67±9.01） pg/ml and （69.89± 5.80） pg/ml vs （10.83± 4.11） pg/ml,（137.68 ±12.28） pg/ml and （72.25± 8.61） pg/ml, respectively. No statistical differences found at 0 h （t = 0.8398, P 〉 0.05） and 24 h （t = 0.6820, P 〉 0.05） between the two groups except that at 6 h （t = 9.653, P 〈 0.01）. The expressions of phospho-IRF3 in HBV group were lower than that in control group. Conclusions The type I interferon secretion and the phospho-IRF3 expression were decreased in murine liver dendritic cells when intervened by HBV.