骨髓间充质干细胞外泌体调节哮喘小鼠Foxp3^＋ Treg/Th17的平衡  

Exosomes from bone marrow mesenchymal stem cells regulate the balance of Foxp3^+ Treg/Th17 in asthmatic mice

作　　者：林颖[1] 胡锦章 颛孙永勋[1] 冉丕鑫[3] 陈瑞[1] 杜雨末[1] 林琳[1] 李建国[1] Lin Ying;Hu Jin-zhang;Zhuansun Yong-xun;Ran Pi-xin;Chen Rui;Du Yu-mo;Lin Lin;Li Jian-guo(Department of Respiratory Medicine,Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation,Sun Yat-Sen Memorial Hospital,Sun Yat-Sen University,Guangzhou 510120,Guangdong Province,China;Ningxi Community Healthy Service Center of Yongning Street,Guangzhou 511300,Guangdong Province,China;the State Key Laboratory of Respiratory Disease,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou 510120,Guangdong Province,China)

机构地区：[1]中山大学孙逸仙纪念医院呼吸内科,广东省恶性肿瘤表观遗传与基因调控重点实验室,广东省广州市510120 [2]广州市增城区永宁街宁西社区卫生服务中心,广东省广州市511300 [3]广州医科大学第一附医院广州呼吸疾病研究所,广东省广州市510120

出　　处：《中国组织工程研究》2018年第17期2637-2643,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然自由申请项目(2015A030313134); 广州市科技计划项目(201604020103)

摘　　要：背景:Th1/Th2失衡是哮喘的基本的病理生理表现,但近来的研究表明哮喘还与调节T细胞(Foxp3^+Treg)/Th17的失衡有关。许多研究表明间充质干细胞的免疫调节作用与其分泌的外泌体相关。目的:观察骨髓间充质干细胞外泌体注射对哮喘小鼠气道炎症及Foxp3^+Treg细胞、Th17细胞及支气管肺泡灌洗液中细胞因子的影响。方法:将27只SPF级BALB/c小鼠分为3组:正常对照组、哮喘模型组、外泌体处理组。除正常对照组外,其他两组以卵白蛋白致敏和激发的方法建立哮喘动物模型,外泌体处理组在第21天激发的同时尾静脉注射外泌体。最后1次激发后24 h,检测各组小鼠脾中Foxp3^+Treg及Th17占淋巴细胞的比例,检测Foxp3^+Treg中CTLA-4及PD-1表达,检测CD4^+T细胞中p27^(kip1)表达,支气管肺泡灌洗液中炎症细胞总数及分类计数,肺泡灌洗液中白细胞介素4,5,13,10,17及干扰素γ表达,并进行肺组织病理观察。结果与结论:(1)小鼠脾组织中Foxp3^+Treg占淋巴细胞比例及Foxp3^+Treg上CTLA-4和PD-1表达:外泌体处理组高于哮喘模型组(P<0.01);(2)脾组织中Th17细胞占淋巴细胞的比例:哮喘模型组>外泌体处理组>正常对照组(P<0.01);(3)支气管肺泡灌洗液中炎症细胞总数及分类计数:外泌体处理组炎症细胞总数、嗜酸性粒细胞计数,单核巨噬细胞计数显著低于哮喘模型组(P<0.01);(4)肺组织病理变化:正常对照组气道无明显炎症细胞浸润,哮喘模型组气道炎症显著,外泌体组气道炎症明显减轻;(5)支气管肺泡灌洗液中细胞因子水平检测:与哮喘模型组相比,外泌体处理组白细胞介素13和Th17相关的白细胞介素17水平降低(P<0.05,P<0.01),Foxp3^+Treg相关的白细胞介素10水平升高(P<0.01);(6)CD4^+T细胞中p27^(kip1)的表达:外泌体处理组p27^(kip1)的表达高于哮喘模型组;(7)结果表明:骨髓间充质干细胞外泌体可以抑制哮喘小鼠气道炎症反应,逆转哮喘小鼠Foxp3^+TregBACKGROUND：Imbalance of Th1/Th2 immune response is an crucial pathophysiological manifestation of asthma,but recent studies have proved that asthma also has a close correlation with the imbalance of Foxp3^＋Treg/Th17.Accumulating evidence indicate that the immunoregulatory capacity of mesenchymal stem cells are mainly related to exosomes secreted by the cells.OBJECTIVE：To observe the effect of bone marrow mesenchymal stem cell exosomes on Foxp3^＋Treg cells,Th17 T cells and airway inflammation of asthmatic mice as well as cytokines in the bronchoalveolar lavage fluid.METHODS：Twenty-seven BALB/c mice of SPF grade were randomly divided into three groups：normal control group,asthmatic model group,and exosomes group.Except the normal control group,each mouse in the other groups was sensitized by ovalbumin to establish asthma models.In the exosomes group,each mouse was intravenously administrated with exosomes at 21 days of sensitization.At 24 hours after the final administration of ovalbumin,the proportion of Foxp3^＋Treg and Th17 in the sleep of asthmatic mice as well as the expression of CTLA-4and PD-1 in Foxp3^＋Treg cells were detected by flow cytometry.The total number of inflammatory cells,and the number of eosinophils,lymphocytes,monocytes and neutrophils in the bronchoalveolar lavage fluid were counted to analyze the degree of airway inflammation in the combination with pathological observation.We also detected the expression of interleukin-4,5,13,10,17 and interferon-γin the bronchoalveolar lavage fluid as well as p27^kip1 in CD4^＋T cells.RESULTS AND CONCLUSION：（1）The proportion of Foxp3^＋Treg in splenic lymphocytes and the CTLA-4 and PD-1 expression in Foxp3^＋Treg were significantly higher in the exosomes group than the asthmatic model group（P0.01）.（2）The proportion of Th17 in splenic lymphocytes was ranked as follows：asthmatic model groupexosomes groupnormal control group（P0.01）.（3）The total number of inflammatory cells and the number of eosinophils,lymphocytes,ne

关 键 词：哮喘 间充质干细胞 外泌体 组织工程 调节性T细胞 免疫调节 炎症 细胞毒T淋巴细胞抗原4 程序性死亡受体1:细胞周期素依赖性蛋白激酶抑制因子 免疫失衡 

分 类 号：R394.2[医药卫生—医学遗传学]