皮下注射大剂量卵白蛋白诱导小鼠哮喘免疫耐受模型的建立及机制初步研究  

Establishment of mouse model of immune tolerance of asthma through subcutaneous injection of high dose of ovalbumin

作　　者：胡琦[1] 钟世民[1] 汪金玉[2] 范文婷[1] 曾靖[1] 吴雪婷[1] 廖伟[1] 

机构地区：[1]第三军医大学西南医院儿科,重庆400038 [2]第三军医大学西南医院胸心外科实验室,重庆400038

出　　处：《第三军医大学学报》2016年第12期1404-1409,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81170034;81570022);重庆市基础与前沿研究计划项目(CSTC2013jcyi A10121)~~

摘　　要：目的腹部皮下注射大剂量卵白蛋白（ovalbumin,OVA）建立小鼠哮喘免疫耐受模型并对机制进行初步研究。方法选择BALB/c小鼠18只为建模对象,按随机数字表法分为3组：哮喘免疫耐受模型组、哮喘对照组、正常对照组,每组6只。哮喘免疫耐受模型组予10μg OVA于0、7 d腹腔注射致敏,21~25 d持续5 d予1 mg OVA腹部皮下注射诱导免疫耐受,35、36、37 d予1%OVA雾化激发;哮喘对照组除了省去21~25 d OVA皮下注射诱导免疫耐受外,其余同哮喘免疫耐受模型组;正常对照组使用等量生理盐水致敏及激发。末次激发24 h内检测气道反应性,收集支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）计数细胞总数及细胞分类计数,ELISA法检测血清OVA特异性Ig E、BALF中IL-4、IFN-γ、IL-10的表达,HE染色观察肺组织病理改变,流式细胞仪检测各组小鼠外周血Treg细胞占CD4＋T淋巴细胞百分率情况。结果哮喘免疫耐受模型组气道反应性、BALF中嗜酸性粒细胞计数、IL-4水平及血清OVA特异性Ig E水平明显低于哮喘对照组,差异有统计学意义（P〈0.05）;BALF中IL-10明显高于哮喘对照组（P〈0.05）;而BALF中IFN-γ水平与哮喘对照组差异无统计学意义;同时HE染色发现哮喘免疫耐受模型组肺组织炎症较哮喘对照组明显减轻;流式细胞技术检测发现外周血Treg细胞百分比明显高于哮喘对照组,差异有统计学意义（P〈0.05）。结论予致敏小鼠腹部皮下注射大剂量OVA可成功诱导小鼠哮喘免疫耐受形成,Treg及免疫抑制因子IL-10参与了免疫耐受的形成。Objective To establish a mouse model of immune tolerance of asthma through subcutaneous injection with high dose of ovalbumin（ OVA） in the abdomen and investigate its underlying mechanism. Methods With a random number table method,18 female BALB / c mice were divided into 3groups： asthmatic immune tolerance model group,asthma control group and normal control group,with 6 mice in each group. The mice in the model group and asthma control group were sensitized with 10 μg OVA by intraperitoneal injection in the abdomen on day 0 and day 7. The mice in the model group induced immune tolerance with 1 mg OVA by subcutaneous injection in the abdomen everyday from day 21 to day 25. Both the model group and asthma control group were challenged with 1% OVA on day 35,day 36 and day 37. The mice in the normal control group was sensitized and challenged with the equal amount of saline. The airway reactivity was detected at 24 h after the last challenge. The enhanced pause（ Penh） was measured to evaluate the airway responsiveness with a noninvasive lung functional instrument. Bronchoalveolar lavage fluid（ BALF）was collected to count the total cells and eosinophils,and the cytological studies were conducted. The OVAspecific Ig E in peripheral blood,and the IL-4,IFN-γ and IL-10 in BALF were detected by enzyme-linked immunosorbent assay（ ELISA）. The lung tissues were obtained to perform histological analysis by HE staining. The percentage of regulatory T cells in peripheral blood was calculated by flow cytometry. Results The airway responsiveness,total cells and eosinophils in BALF of the model group were significantly decreased as compared with the asthma control group（ P 0. 05）. The OVA-specific Ig E in peripheral blood and IL-4 in BALF were significantly lower in the model group than that in the asthma control group（ P 0. 05）,and on the contrary,the levels of IL-10 were significantly higher（ P 0. 05）. The levels of IFN-γ had no significant difference. The inflammation of lung tissues was allevia

关 键 词：卵白蛋白 小鼠模型 支气管哮喘 免疫耐受 

分 类 号：R332[医药卫生—人体生理学] R341[医药卫生—基础医学]