多房棘球蚴感染小鼠辅助Th17细胞,转录因子RORγτ及相关细胞因子的实验研究

Alteration and significance of T helper cell 17,transcription factor RORγτ,and IL-17 in mice infected with Echinococcus multilocularis

目的探讨多房棘球蚴感染小鼠脾脏细胞中辅助性T细胞17比例,转录因子RORγτ和IL-17因子的表达及其意义。方法选用6~8周龄BALB/c小鼠共30只,随机分为多房棘球蚴感染组（Em）、多房棘球蚴感染阿苯达唑治疗组（Em＋Albendazole,Em＋ABZ）和健康对照组（healthy controls,HC）,每组10只。通过腹腔注射法建立泡型包虫病小鼠模型。Em＋ABZ治疗组给予灌胃阿苯达唑治疗（100μl/d,35d）通过流式细胞术检测小鼠脾脏细胞中辅助性T17细胞（CD4＋IL-17＋Th17细胞/CD4＋T细胞）比例,采用实时荧光定量PCR检测脾脏细胞中维甲酸孤独受体（retinoid acid orphan receptor-γτ,RORγτ）mRNA的表达;采用酶联免疫吸附试验（ELISA）检测IL-17水平。结果 CD4＋IL-17＋Th17细胞/CD4＋T细胞比例,Em组为（2.10±1.00）%,与HC组（0.85±0.71）%比较,差异有统计学意义（F=3.07,t=2.66,P〈0.01）。Em＋ABZ组为（1.54±1.19）%,与HC组和Em组比较,差异无统计学意义（F=3.07,P〉0.05）;脾脏细胞RORγτmRNA在Em组中的表达（6.1×10-4±5.8×10-4）高于在Em＋ABZ组（6.0×10-4±3.5×10-4）和HC组（3.4×10-4±2.4×10-4）中的表达,但差异无统计学意义（F=0.66,P〉0.05）。Em＋ABZ组与HC组比较,差异亦无统计学意义（F=0.66,P〉0.05）。脾脏细胞培养上清液中IL-17A,Em组为（10.71±1.85）ng/ml,明显高于Em＋ABZ组[（5.58±3.56）ng/ml]和HC组[（7.31±1.22）ng/ml],差异有统计学意义（F=9.82,t1=3.12,t2=5.57,P〈0.01）。Em＋ABZ组与HC组比较差异无统计学意义（F=9.82,t=0.84,P〉0.05）。结论 Th17细胞参与BALB/c小鼠多房棘球蚴感染免疫和炎性应答过程,阿苯达唑治疗能够减轻免疫损伤和炎症反应。Th17细胞及IL-17A可能是潜在的多房棘球蚴病新的分子免疫干预靶点。

Objective To explore the alteration and significance of T helper cell 17,transcription factor RORγτ,and IL-17 in BALB/c mice infected with Echinococcus multilocularis. Methods Thirty 6-8week-old BALB/c mice were randomly divided into three groups：mice infected with E.multilocularis（n=10）,mice infected with E.multilocularis＋treated with albendazole（ABZ）（n=10）,and healthy controls（HCs,n=10）.A model of alveolar echinococcosis was created by intraperitoneally infecting mice with E.multilocularis.Mice infected with E.multilocularis＋treated with ABZ were administered ABZ（100ul/d for 35days）via a gastric tube.The proportion of Th17 cells in the spleen was determined using flow cytometry,expression of RORγτmRNA was determined using real-time PCR,and levels of IL-17 in splenic cell supernatant were detected using ELISA. Results The ratio of CD4＋IL-17A＋Th17cells/CD4＋T cells in the spleen was significantly higher in mice infected with E.multilocularis（2.10±1.00%）than in the HCs（0.85±0.71%,F=3.07,t=2.66,P〈0.01）.The ratio of CD4＋IL-17A＋Th17cells/CD4＋T was higher in mice infected withE.multilocularis＋treated with ABZ（1.54±1.19%）than in HCs but lower than that in mice infected with E.multilocularis,although the difference was not statistically significant（F=3.07,P〉0.05）.The level of expression of the transcription factor RORγτin splenic cells was higher in mice infected with E.multilocularis（6.1×10-4±5.8×10-4）than in HCs（3.4×10-4±2.4×10-4）and in mice infected with E.multilocularis＋treated with ABZ（6.0×10-4±3.5×10-4）,but the difference was not statistically significant.The level of RORγτexpression was higher in mice infected with E.multilocularis＋treated with ABZ than in HCs,but the difference was not statistically significant（F=0.66,P〉0.05）.The level of IL-17 Aexpression in splenic cell supernatant was significantly higher in mice infected with E.multilocularis（10.71±1.85ng/ml）than in mice infected with E.multilocularis＋treated with ABZ（5.58±3.56ng/ml）and in HCs（7.31±1.22ng/ml）,and the difference was statistically significant（F=9.82,t1=3.12,t2=5.57,P〈0.01）.However,the level of IL-17 Aexpression did not differ significantly in mice infected with E.multilocularis＋treated with ABZ and HCs（F=9.82,t=0.84,P〉0.05）. Conclusion Th17 cells are actively involved in immunity from infection and inflammatory response in a murine model of alveolar echinococcosis.ABZ reduced the number of Th17 cells and related cytokine levels,possibly alleviating immunopathological injury and inflammation.Th17 cells and related cytokines may be a potential therapeutic molecular target for alveolar echinococcosis.