Treg细胞抑制慢性中耳炎模型小鼠中耳炎症的进展  

Treg cells inhibits the progression of middle ear inflammation in mice with chronic otitis media

作　　者：王丹 杨启梅[2] 刘晓娜 陈菁华 WANG Dan;YANG Qimei;LIU Xiaona;CHEN Jinghua(Department of Otolaryngology,North-West Women and Children′s Hospital,Xi′an 710061,China;Department of Otorhinolaryngology-Head and Neck Surgery,Shaanxi Provincial People′s Hospital)

机构地区：[1]西北妇女儿童医院耳鼻喉科,西安710061 [2]陕西省人民医院耳鼻咽喉头颈外科

出　　处：《山西医科大学学报》2024年第2期205-213,共9页Journal of Shanxi Medical University

基　　金：陕西省重点研发计划项目(2023-YBSF-081)。

摘　　要：目的探究慢性中耳炎(chronic otitis media,COM)小鼠中Treg细胞与炎症发展之间的关系。方法采用不可分型流感嗜血杆菌(NTHi)腹腔注射和中耳咽鼓管(ET)阻断的方式诱导构建慢性中耳炎小鼠模型(模型组),选择健康BALB/c小鼠作为对照组。分别在建模后第3,14,28,56天分离收集小鼠中耳积液和中耳黏膜组织,通过苏木精-伊红(HE)染色检测MEM病理变化,采用免疫组织化学染色评估CD4^(+)和Foxp3^(+)T细胞水平,ELISA和Western blot分别检测小鼠中耳积液和中耳黏膜组织中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-10和转化生长因子(TGF-β)水平,流式细胞术分析CD4^(+)CD25^(+)Foxp3^(+)细胞比例。另取COM模型小鼠分为PBS组和抗-CD25组。抗-CD25组小鼠腹腔注射抗CD25 mAb模拟体内Treg耗竭,PBS组小鼠腹腔注射等量PBS溶液,第56天收集小鼠中耳积液、中耳黏膜组织、脾脏和颈部淋巴结组织,检测分析脾脏、颈部淋巴结和中耳黏膜组织中CD4^(+)CD25^(+)Foxp3^(+)细胞比例,中耳黏膜组织中炎性因子水平,以及中耳积液中细菌水平。结果HE结果显示,对照组小鼠中耳黏膜组织正常,模型组小鼠中耳黏膜组织出现炎性细胞浸润,细菌数量、炎性细胞数量和黏膜厚度均增加(P<0.05)。与对照组比较,模型组小鼠中耳黏膜组织中CD4^(+)CD25^(+)Foxp3^(+)细胞比例升高(P<0.05),中耳积液和中耳黏膜组织中炎性因子IL-1β、TNF-α、IL-10和TGF-β的水平均升高(P<0.01)。与PBS组比较,抗-CD25组小鼠中耳黏膜组织、脾脏和颈部淋巴结组织中CD4^(+)CD25^(+)Foxp3^(+)细胞比例均降低(P<0.05),中耳积液中细菌数和中耳黏膜组织中炎性因子IL-1β、TNF-α、IL-10和TGF-β的水平均降低(P<0.01)。结论Treg细胞在慢性中耳炎的炎症平衡维持中具有重要作用,抑制炎症的进展,但过度的Treg细胞抑制了宿主对细菌的清除作用。Objective To explore the relationship between Treg cells and inflammation development in chronic otitis media(COM)mice.Methods COM mouse model was induced by intraperitoneal injection of non-typeable Haemophilus influenzae(NTHi)and blocking of eustachian tube(ET)in the middle ear,and healthy BALB/c mice were used as control group.Middle ear effusion(MEE)and middle ear mucosal(MEM)tissue of mice were collected at day 3,14,28 and 56 after modeling.The pathological changes of MEM tissue were detected by hematoxylin-eosin(HE)staining.The CD4^(+)and Foxp3^(+)T cell levels were evaluated by immunohistochemical staining.The levels of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β),IL-10 and transforming growth factor(TGF-β)in MEE and MEM tissue of mice were detected by enzyme-linked immunosorbent assay(ELISA)and Western blot,respectively.The proportion of CD4^(+)CD25^(+)Foxp3^(+)cells was analyzed by flow cytometry.COM model mice were divided into PBS group and anti-CD25 group.The mice in anti-CD25 group were intraperitoneally injected with anti-CD25 mAb to simulate Treg depletion in vivo,and the mice in PBS group were intraperitoneally injected with the same amount of PBS solution.The MEE,MEM tissue,spleen and cervical lymph nodes of the mice were collected at day 56.The proportion of CD4^(+)CD25^(+)Foxp3^(+)cells in spleen,cervical lymph nodes and MEM tissue,and the level of inflammatory factors in MEM and the level of bacteria in MEE were detected and analyzed.Results HE results showed that MEM of mice in control group was normal,while the inflammatory cell infiltration occurred in MEM tissue of mice in model group,and the numbers of bacteria and inflammatory cells and the thickness of MEM were increased(P<0.05).Compared with control group,the proportion of CD4^(+)CD25^(+)Foxp3^(+)cells in MEM tissues of mice was increased in model group(P<0.05),and the levels of inflammatory factors IL-1β,TNF-α,IL-10 and TGF-βin MEE and MEM tissues were increased(P<0.01).Compared with PBS group,the proportion of CD4^(+)C

关 键 词：慢性中耳炎 不可分型流感嗜血杆菌(NTHi) Treg细胞 中耳积液 中耳黏膜组织 

分 类 号：R764.221[医药卫生—耳鼻咽喉科]