卡介菌-多糖核酸对哮喘小鼠淋巴细胞CD4CD25+调节性T细胞的影响  

Effects of bacillus calmette-guerin polysaccharide nucleotide on CD4+ CD25+ Treg in lymph of asthmatic mouse model

作　　者：姜华 赖克方[2] 南岩东 金发光 Jiang Hua;Lai Kefang;Nan Yandong;Jin Faguang(Department of Pulmonary and Critical Care Medicine,Tangdu Hospital,Air Force Military Medical University,Xi an 710038,China;The First Affiliated Hospital of Guangzhou Medical University,Guangzhou Institute of Respiratory Health,Guangzhou 510120,China)

机构地区：[1]空军军医大学唐都医院呼吸与危重症医学科,西安710038 [2]广州医科大学第一附属医院广州呼吸健康研究院,510120

出　　处：《国际呼吸杂志》2019年第12期881-887,共7页International Journal of Respiration

基　　金：空军军医大学唐都医院科技创新发展基金(2016JCYJ015).

摘　　要：目的 探讨卡介菌多糖核酸(BCG-PSN)对哮喘小鼠淋巴细胞CD4+CD25+调节性T细胞(CD4+CD25+T reg细胞)数量及功能的影响.方法 选择Balb/c小鼠,以卵白蛋白致敏激发建立小鼠哮喘模型,分为对照组、哮喘组、BCG-PSN干预组.BCG-PSN剂量为20μg/只,体积为60μl,在致敏前7 d腹腔注射干预,对照组同时给予相同剂量的生理盐水作对照,末次激发后48 h对小鼠进行有创肺功能检测,以乙酰甲胆碱各浓度激发时肺阻力(RL)表示.并收集支气管肺泡灌洗液(BALF),涂片后HE染色,计数其中的嗜酸粒细胞(EOS),取各组小鼠脾脏淋巴细胞,体外培养72 h后流式细胞仪检测CD4+CD25+T reg细胞/CD4+T细胞,Real-time PCR检测脾脏淋巴细胞foxp3及CTLA-4基因的表达量,酶联免疫吸附试验法检测脾脏淋巴细胞上清液中白细胞介素-10(IL-10)、转化生长因子β(TGF-β)的含量.结果 (1)BCG-PSN干预可明显降低哮喘小鼠的气道反应性和气道炎症.(2)哮喘组CD4+CD25+T reg细胞/CD4+T细胞比例(3.71±2.20)% 明显低于正常组(6.44±1.16)%(P<0.01),BCG-PSN组(6.75±1.63)% 与哮喘组相比差异有统计学意义(P<0.01).(3)哮喘组小鼠脾脏淋巴细胞foxp3 Mrna、CTLA-4 Mrna的表达量明显低于正常组,BCG-PSN干预可显著提高foxp3 Mrna、CTLA-4 Mrna的表达.(4)哮喘小鼠脾脏淋巴细胞上清中IL-10、TGF-β的含量明显少于正常组.BCG-PSN组IL-10含量为(210.32±88.23)ng/L,显著高于哮喘组(114.41±73.58)ng/L(P<0.05).BCG-PSN组TGF-β含量为(487.66±32.57)ng/L,显著高于哮喘组(97.76±23.58)ng/L(P<0.01).结论 BCG-PSN可增加CD4+ CD25+ Treg细胞数量及功能 ,具体机制可能通过促进IL-10、TGF-β等抑制性细胞因子的分泌 ,及CTLA-4介导的细胞-细胞接触抑制机制诱导免疫耐受 ,从而降低哮喘小鼠的气道反应性和气道炎症.Objective To investigate the effect of bacillus calmette-guerin polysaccharide nucleotide (BCG-PSN) on quantity and function of CD4+CD25+ Treg in lymph of asthmatic mouse model .Methods Balb/c mice were sensitized by ovalbumin to establish the asthmatic mouse model .The mice of control group received saline at every time point .A pretreatment of 20 μg/60 μl of BCG-PSN by intraperitoneal injection were done at 7 days before the first sensitization .Bronchial hyper responsiveness (BHR) ,eoxinophil (EOS)% in broncho alveolar lavage fluid (BALF) were examined 48 hours after the final challenge .Propotion of spleen CD4+CD25+ Treg cells/CD4+ cells was measured by flow cytometry . Foxp3 and CTLA-4 expression in spleen lymphocytes was analyzed with RT and real-time PCR .IL-10 ,TGF-β levels in the cultures spleen lymphocytes supernatant were examined with ELISA .Results (1)BCG-PSN intervention significantly decreased airway inflammation and airway hyperresponsiveness in asthmatic mice .( 2) The percentage of CD4+CD25+ Treg cells/CD4+ (3 .71 ± 2 .20)% in the asthma group was significantly lower than that in the normal group ( 6 .44 ± 1 .16)% ( P <0 .01) ,there was significant difference between the BCG-PSN group (6 .75 ± 1 .63)% and the asthma group .(3)The expression of foxp3 Mrna and CTLA-4 Mrna in spleen lymphocytes of asthmatic mice was significantly lower than that in normal mice .( 4) The intervention of BCG-PSN significantly increased the expression of CTLA-4 Mrna and foxp3 Mrna .The levels of IL-10 and TGF-βin the spleen lymphocyte supernatant of asthmatic mice were significantly less than those in normal mice .The level of IL-10 in BCG-PSN group was ( 210 .32 ± 88 .23) ng/L which was significantly higher than that in asthma group (114 .41 ± 73 .58) ng/L ,and the level of TGF-β in BCG-PSN group (487 .66 ± 32 .57) ng/L was significantly higher than that in the asthma group ( 97 .76 ± 23 .58) ng/L ( P <0 .01) .Conclusions BCG-PSN can increase immune tolerance on asthmatic mice by promoted the quatity and

关 键 词：卡介菌多糖核酸 哮喘 支气管 调节性T细胞 

分 类 号：R5[医药卫生—内科学]