人脐带间充质干细胞源胞外囊泡对屋尘螨诱导的哮喘小鼠气道炎症的影响

Effect of human umbilical cord mesenchymal stem cell-derived extracellular vesicles on house dust mite-induced airway inflammation in asthmatic mice

目的:探讨人脐带间充质干细胞源胞外囊泡(human umbilical cord mesenchymal stem cells-derived extracellular vesicles, hUCMSC-EVs)对屋尘螨(house dust mite, HDM)诱导的哮喘小鼠气道炎症的影响。方法:超高速离心法提取hUCMSC-EVs;透射电镜观察hUCMSC-EVs形态;蛋白质印迹法检测胞外囊泡标志物肿瘤易感基因101(tumor susceptibility gene 101,TSG101)和热休克蛋白70(heat shock protein 70,HSP70)表达。将15只雌性BALB/C小鼠随机均分为3组,每组5只,分别为对照组(不做任何处理)、HDM组(HDM处理)和HDM+EVs组(HDM+hUCMSC-EVs处理);苏木精-伊红(HE)和过碘酸-雪夫(PAS)染色法分别观察各组小鼠肺脏气道炎性细胞浸润和杯状细胞化生;收集各组小鼠支气管肺泡灌洗液(BALF)并对总细胞和嗜酸性粒细胞进行计数;ELISA法检测BALF中IL-4和IL-13含量;蛋白质印迹法检测肺脏中E-钙黏蛋白(E-cadherin)和闭锁连接蛋白1(ZO-1)表达。将人支气管上皮BEAS-2B细胞株分为对照组和EVs组(DIO标记的hUCMSC-EVs处理),共聚焦显微镜观察细胞对DIO标记的hUCMSC-EVs的内吞情况。另将BEAS-2B细胞分为3组:对照组、HDM组(HDM处理)、HDM+EVs组(HDM+hUCMSC-EVs处理),蛋白质印迹法检测细胞中E-cadherin和ZO-1表达。结果:hUCMSC-EVs呈杯托样膜性结构且表达HSP70和TSG101。与HDM组相比,HDM+EVs组小鼠气道炎性评分、PAS评分、BALF中总细胞和嗜酸性粒细胞数、IL-4和IL-13含量均显著下调(P均<0.01),而肺脏组织中E-cadherin和ZO-1表达明显增加(P<0.05)。体外实验显示hUCMSC-EVs可被人支气管上皮BEAS-2B细胞内吞;与HDM组相比,HDM+EVs组BEAS-2B细胞中E-cadherin和ZO-1表达明显增加(P<0.01或<0.05)。结论:hUCMSC-EVs可显著改善HDM诱导的小鼠气道周围炎性细胞浸润和气道杯状细胞化生。

Objective:To investigate the effect of human umbilical cord mesenchymal stem cells-derived extracellular vesicles(hUCMSC-EVs)on house dust mite(HDM)-induced airway inflammation in asthmatic mice.Methods:hUCMSC-EVs was extracted by ultra-high speed centrifugation;morphological characteristics of hUCMSC-EVs were observed by transmission electron microscopy.EVs markers,tumor susceptibility gene 101(TSG101)and heat shock protein 70(HSP70),were detected by Western blotting.Fifteen female BALB/C mice were randomly divided into 3 groups,5 mice in each group,including control group(without any treatment),HDM group(HDM treatment)and HDM+EVs group(HDM+hUCMSC-EVs treatment).Hematoxylin and eosin(HE)and periodic acid-Schiff(PAS)staining were used to observe the inflammatory cell infiltration and goblet cell hyperplasia of lung airway in each group.Bronchoalveolar lavage fluid(BALF)was collected,and the numbers of total cells and eosinophils were counted.The contents of IL-4 and IL-13 in BALF were detected by ELISA.The expression of E-cadherin and atresia connexin 1(ZO-1)in the lungs was detected by Western blotting.Human bronchial epithelial BEAS-2B cell lines were divided into control group and EVs group(DIO-labeled hUCMSC-EVs treatment).Confocal microscopy was used to observe the endocytosis of DIO-labeled hUCMSC-EVs cells.BEAS-2B cells were divided into three groups:control group,HDM group(HDM treatment),and HDM+EVs group(HDM+hUCMSC-EVs treatment),and the protein expression of E-cadherin and ZO-1 in the cells was detected by Western blotting.Results:hUCMSC-EVs showed a circular membrane structure and expressed HSP70 and TSG101.Compared with HDM group,airway inflammatory score,PAS score,the number of total cells and eosinophils in BALF,the contents of IL-4 and IL-13 in BALF were significantly decreased in HDM+EVs group(all P<0.01),whereas E-cadherin and ZO-1 expressions in lung tissues were markedly increased(P<0.05).In vitro experiments showed that hUCMSC-EVs could be endocytosed by human bronchial epithelial cell line BEAS-2B.Compared with HDM group,the expressions of E-cadherin and ZO-1 were significantly increased in HDM+EVs group(P<0.01 or<0.05).Conclusion:hUCMSC-EVs could ameliorate HDM-induced peri-airway inflammatory cell infiltration and airway goblet cell hyperplasia in mice.