Th17/Treg细胞介导的分泌性中耳炎发病机制研究  

Th17/Treg Cells Mediated Pathogenesis in Otitis Media with Effusion

作　　者：陈沛维 赵春丽[1] 王丹妮[1] 杨劲松[1] 李洁[2] 赵守琴[1] CHEN Peiwei;ZHAO Chunli;LI Jie;YANG Jinsong;ZHAO Shouqin(Department of Otorhinolaryngology-Head and Neck Surgery,Beijing Tongren Hospital,Capital Medical University,Beijing 100730;Department of Otorhinolaryngology-Head and Neck Surgery,Fuxing Hospital,Capital Medical University,Beijing 100038)

机构地区：[1]首都医科大学附属北京同仁医院院耳鼻咽喉头颈外科,北京100730 [2]首都医科大学附属复兴医院耳鼻咽喉头颈外科,北京100038

出　　处：《中华耳科学杂志》2020年第3期431-437,共7页Chinese Journal of Otology

基　　金：北京市自然科学基金(7162045,7202032)资助。

摘　　要：目的研究分泌性中耳炎(otitis media with effusion,OME)中Th17/调节性T细胞(tregulatory,Treg)及其相关细胞因子、特异性转录因子的变化,深入探索OME免疫学发病机制。方法同批次健康雄性SD大鼠,随机分为对照组(20只,40耳),实验组(20只,40耳)。实验组大鼠采用卵清蛋白腹腔致敏及鼓膜穿刺耳内致敏2次制成变应原诱发的变态反应相关的OME SD大鼠模型,对照组予以空白对照处理,基于内镜局部积液征、组织学及电镜观察验证建模成功;从免疫细胞、分子和基因三个水平检测、比较OME组及对照组大鼠外周血及中耳局部变化:通过流式细胞技术(flow cytometry,FCM)、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)和逆转录-聚合酶链反应(reverse transcription-polymerase chain Reaction,RT-PCR)分别检测外周血Th17细胞和CD4^+CD25^+Foxp3^+Treg细胞、相关细胞因子白细胞介素-17(interleukin 17,IL-17)和转化生长因子-β(transforming growth factor beta,TGF-β);中耳局部对应特异性转录因子维甲酸相关孤核受体γt(retinoid-related orphan receptorγt,ROR-γt)、叉状头转录因子3(forkhead box protein 3,Foxp3)m RNA表达水平在OME组及对照组的变化。结果基于内镜、组织学及电镜评估,成功建立免疫相关的OME大鼠模型;外周血TGF-β浓度测定示实验组:95.79±21.60 pg/ml,对照组:72.49±19.75 pg/ml,实验组较对照组显著升高(P=0.005<0.05),差异有统计学意义;外周血IL-17浓度测定实验组与对照组差异无统计学意义(P>0.05);实验组与对照组外周血Th17细胞、CD4^+CD25^+Foxp3^+Treg细胞及两者比值测定差异无统计学意义(P>0.05)。中耳局部检测示实验组中耳黏膜局部Foxp3mRNA表达OME组较对照组增高(P=0.0036<0.05);RORγt m RNA表达两者差异无统计学意义(P=0.1069>0.05)。结论OME的免疫相关发病机制之一可能是全身免疫反应与局部免疫反应的共同作用的结果,以中耳局部免疫反应为主;TGF-β�Objective To study changes of Th17/Regulatory T cells(Treg),related cytokines and specific transcription factors in otitis media with effusion(OME)in rats in relation to immunological pathogenesis of otitis media with effusion.Methods Healthy male Sprague-Dawley rats were randomly divided into a control group(n=22,44 ears)and an OME group(n=30,60 ears).Allergy-related OME was modeled by peritoneal ovalbumin sensitization followed by two sessions of trans-tympanic membrane sensitization.Rats in the control group received sham treatment.Modeling success was confirmed by signs of effusion on ear endoscopy,as well as histology and electron microscopy evidence.Animals were sacrificed on the 18th day after modeling and specimens collected for examination.Imunocellular,molecular and genetic indicators in peripheral blood and the middle ear were compared between the OME and control groups.Serum Th17 cells and CD4+CD25+Foxp3+Treg cells were detected by flow cytometry(FCM).Relevant cytokine interleukin 17(IL-17)and transforming growth factor beta(TGF-β)were detected by enzyme-linked immunosorbent assay(ELISA).Expression levels of retinoid-related orphan receptorγt(ROR-γt)mRNA and forkhead box protein 3(Foxp3)mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Modeling of immune-mediated OME was successful as verified by ear endoscopy,histology and electron microscopy assessment.Serum TGF-βlevel was 95.79±21.60 pg/ml in the OME group and 72.49±19.75 pg/ml in the control group(P=0.005<0.05).There was no statistically significant difference in serum IL-17 or in the ratio of Th17/CD4+CD25+Foxp3+Treg cells between the two groups(P>0.05).The level of Foxp3 mRNA expression in middle ear mucosa was higher in the OME group than in the control group(P=0.0036<0.05),while there was no significant difference in RORγt mRNA expression(P=0.1069>0.05).Conclusion One of the may be a Combined systemic and local immune response may be a immune mechanism for OME with local immune response in the tympani

关 键 词：分泌性中耳炎 TREG细胞 Th17细胞 TGF-β Foxp3 mRNA 

分 类 号：R764[医药卫生—耳鼻咽喉科]