多房棘球蚴感染对胶原蛋白诱导小鼠自身免疫性关节炎的影响研究

The effects of infection with Echinococcus multilocularis on autoimmune arthritis induced by collagen in C57 mice

目的探讨多房棘球幼（E.m）感染所致的免疫应答偏倚对实验性类风湿性关节炎（CIA）的免疫调节作用。方法分别建立雌性C57小鼠胶原性关节炎模型（CIA组）、多房棘球蚴感染模型（E.m组）和多房棘球蚴感染后类风湿性关节炎诱导模型（E.m＋CIA组）,利用实时荧光定量RT-PCR和酶联免疫吸附试验（ELISA）检测小鼠足爪组织、外周血及脾脏细胞中Th17细胞相关因子及转录因子水平,实验设阴性对照组（Control组）。组间比较采用非参数秩和检验两个独立样本比较法（M-W法）;采用Spearman线性相关性分析方法分析细胞因子及转录因子之间的相关关系。结果外周血及脾脏细胞中IL-17A、IL-1β、IFN-γ水平在E.m＋CIA组18.93（14.90-20.53）pg/ml、18.93（14.90-20.53）pg/ml,4.18（1.79-4.45）pg/ml、4.182（1.794-4.446）pg/ml,14.79（0.64-29.38）pg/ml、14.79（0.64-29.38）pg/ml,显著低于CIA组16.47（15.41-19.46）pg/ml、16.47（15.41-19.46）pg/ml,161.60（125.40-222.20）pg/ml、367.2（240.4-550.3）pg/ml,29.89（19.93-58.95）pg/ml、29.89（19.93-58.95）pg/ml（P〈0.05或P〈0.01）;E.m＋CIA组IL-4、IL-6等抗炎相关因子水平11.68（11.00-12.85）pg/ml、11.68（11.00-12.85）pg/ml,450.40（292.80-697.50）pg/ml、450.40（292.80-645.60）pg/ml,较其他组显著升高（P〈0.05或P〈0.01）。小鼠足爪中IL-17A及RORrt mRNA相对表达量在E.m＋CIA组13.29（5.38-18.93）,1.07（0.78-6.99）较其他组降低。结论 E.m感染诱导的Th2优势应答可使CIA不能有效启动致病性Th1免疫应答,抑制CIA小鼠过度的Th17应答,从而减轻RA炎症损伤。

Objective To explore the effects of immune response bias caused by infection with Echinococcus multilocularis on immune system regulation as a result of experimentally induced rheumatoid arthritis. Methods Thirty-two 5-6week old C57 mice were randomly divided into four groups（n=8in each group）：（1）negative control group（control）,（2）E.multilocularis group：healthy mice were inoculated with E.multilocularis,（3）CIA group：rheumatoid arthritis was induced in healthy mice using collagen,（4）E.multilocularis＋CIA group：healthy mice were infected with E.multilocularis and then rheumatoid arthritis was induced with collagen.The levels of Th17 cells and Th17-related cytokines in the spleen and peripheral blood and the tissue of the paw were detected using RT-PCR and ELISA.Groups were compared using a non-parametric rank sum test for two independent samples.Spearman linear correlation analysis was used to study the relationship between cytokines and transcription factors. Results In the E.multilocularis＋CIA group,the level of IL-17 Ain peripheral blood was 18.93pg/ml（range：14.90-20.53pg/ml）and that in spleen cells was 18.93pg/ml（range：14.90-20.53pg/ml）,the level of IL-1βin peripheral blood was 4.18pg/ml（range：1.79-4.45pg/ml）and that in spleen cells was 4.182pg/ml（range：1.794-4.446pg/ml）,and the level of IFN-γin peripheral blood was14.79pg/ml（range：0.64-29.38pg/ml）and that in spleen cells was 14.79pg/ml（range：0.64-29.38pg/ml）.In the CIA group,the level of IL-17 Ain peripheral blood was 16.47pg/ml（range：15.41-19.46pg/ml）and that in spleen cells was 16.47pg/ml（range：15.41-19.46pg/ml）,the level of IL-1βin peripheral blood was 161.60pg/ml（range：125.40-222.20pg/ml）and that in spleen cells was 367.2pg/ml（range：240.4-550.3pg/ml）,and the level of IFN-γin peripheral blood was 29.89pg/ml（range：19.93-58.95pg/ml）and that in spleen cells was 29.89pg/ml（range：19.93-58.95pg/ml）.The levels of IL-17 A,IL-1β,and IFN-γin peripheral blood and spleen cells were lower in the E.multilocularis＋CIA group than those in the CIA group.In the E.multilocularis＋CIA group,the level of the anti-inflammatory cytokine IL-4in peripheral blood was 11.68pg/ml（range：11.00-12.85pg/ml）and that in spleen cells was11.68pg/ml（range：11.00-12.85pg/ml）and the level of the anti-inflammatory cytokine IL-6in peripheral blood was450.40pg/ml（range：292.80-697.50pg/ml）and that in spleen cells was 450.40pg/ml（range：292.80-645.60pg/ml）.The levels of anti-inflammatory cytokines like IL-4and IL-6were higher in the E.multilocularis＋CIA group than those in the CIA group.The level of IL-17 A mRNA expression in the tissue of the paw was 23.94（range：17.04-39.50）in the CIA group and 13.29（range：5.38-18.93）in the E.multilocularis＋CIA group,so the level of IL-17 A mRNA expression decreased significantly（P〈0.05）.The level of RORrt mRNA expression was 1.07（range：0.78-6.99）in the E.multilocularis＋CIA group and 1.56（range：1.23-6.71）in the CIA group,so the level of RORrt mRNA expression did not differ significantly between the two groups（P〉0.05）. Conclusion Infection with E.multilocularis induced a Th2-dominant immune response but CIA precluded the effective initiation of a pathogenic Th1 immune response.In addition,infection with E.multilocularis markedly inhibited an excessive Th17 immune response in mice with CIA.These two findings will help to reduce the inflammatory damage of rheumatoid arthritis.