外泌体源性miRNA-21-5p/Smad7在石英粉尘致大鼠肺纤维化中的作用  

Role of exosome-derived miRNA-21-5p/Smad7 in quartz dust-induced pulmonary fibrosis in rats

作　　者：卢炀 丁晓慧 王甜甜 徐萌彤 郝嘉瑞 李文静 宋静[1,2,3] LU Yang;DING Xiaohui;WANG Tiantian;XU Mengtong;HAO Jiarui;LI Wenjing;SONG Jing(School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Key Laboratory of Coal Environmental Pathogenicity and Control,Ministry of Education,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Key Laboratory of Pneumoconiosis,National Health Commission,Shanxi Medical University,Taiyuan,Shanxi 030001,China)

机构地区：[1]山西医科大学公共卫生学院,山西太原030001 [2]山西医科大学煤炭环境致病与防治教育部重点实验室,山西太原030001 [3]山西医科大学国家卫健委尘肺病重点实验室,山西太原030001

出　　处：《环境与职业医学》2024年第8期861-866,共6页Journal of Environmental and Occupational Medicine

基　　金：国家卫生健康委员会尘肺重点实验室项目(2020-PT320-005);山西省自然科学基金面上项目(202303021211123)。

摘　　要：[背景]石英粉尘在肺部无法降解,职业生产过程中吸入大量石英粉尘会导致肺纤维化的发生,进而发展为矽肺病。近年来,研究发现外泌体可能通过携带微小核糖核酸(miRNA)参与纤维化疾病的发病过程,但其在矽肺进程中的作用机制仍然有待研究。[目的]研究外泌体源miRNA-21-5p/抗生物皮肤生长因子母体同源蛋白7(Smad7)在石英粉尘致大鼠肺纤维化的中作用。[方法]24只健康雄性SD大鼠被随机分为4组(每组6只):对照4周组、对照16周组和石英4周组、石英16周组。在实验开始时,采用一次性非暴露式气管内染尘的方式分别向石英组和对照组大鼠气管内注入1 mL石英悬液(50 mg·mL^(-1))和1 mL生理盐水。染尘后第4周、16周行肺泡灌洗术获取肺泡灌洗液,采用聚乙二醇(PEG)沉淀法提取灌洗液内的外泌体,透射电子显微镜(TEM)进行形态学的鉴定,纳米追踪分析(NTA)检测外泌体的粒径,免疫印迹法(WB)检测外泌体的标志蛋白CD9和CD63。采用逆转录聚合酶链反应(RT-PCR)法测定外泌体内miRNA-21-5p的表达水平;采用苏木精-伊红染色法(HE)染色及马松染色法(Masson)观察大鼠肺组织损伤及纤维化病变程度;采用羟脯氨酸(HYP)法检测肺组织胶原含量;采用WB检测肺组织Smad7蛋白的表达。[结果]病理染色结果显示,与对照组相比,染尘4周后大鼠肺组织炎细胞浸润,肺泡壁增厚,胶原蛋白增多,染尘16周后出现胶原蛋白沉积和矽结节;与对照组相比,染尘4周、16周时石英组大鼠肺组织中HYP的表达水平均增高(P<0.05);透射电镜下可见外泌体呈经典的茶托样结构,NTA分析显示其平均粒径为95.8 nm,WB检测到外泌体标志蛋白CD9、CD81表达阳性;染尘4周、16周时,与对照组相比,石英组大鼠肺泡灌洗液内外泌体源性miRNA-21-5p表达均增高(P<0.05),肺组织Smad7蛋白表达量均降低(P<0.05)。[结论]外泌体源性miRNA-21-5p和Smad7可能参与了石英粉尘致大�[Background]Quartz dust cannot be degraded in the lungs,and inhalation of a large amount of quartz dust in the occupational production process will lead to the occurrence of pulmonary fibrosis,and then develop into silicosis.In recent years,studies have found that exosomes may be involved in the pathogenesis of fibrotic diseases by carrying microribonucleic acid(miRNA),but the mechanism of their actions in silicosis still needs to be studied.[Objective]To investigate the role of exosome-derived miRNA-21-5p/mothers against decapentaplegic homolog 7(Smad7)in quartz dust-induced pulmonary fibrosis in rats.[Methods]Twenty-four healthy male SD rats were randomly divided into four groups(six rats in each group):control 4-week group,control 16-week group,quartz 4-week group,and quartz 16-week group.At the beginning of the experiment,1 mL of quartz suspension(50 mg·mL^(-1))and 1 mL of normal saline were injected into the trachea of rats in the quartz group and the control group,respectively,by means of one-time non-exposure intratracheal dust staining.Alveolar lavage was performed at the 4th and 16th weeks after dust staining,the exosomes in lavage solution were extracted by polyethylene glycol(PEG)precipitation,morphological identification was conducted by transmission electron microscopy(TEM),particle size of exosomes was detected by nano-tracking analysis(NTA),and the marker proteins CD9 and CD63 of exosomes were detected by Western blotting(WB).The expression of miRNA-21-5p in exosomes was determined by reverse transcription polymerase chain reaction(RT-PCR).The degree of lung tissue injury and fibrosis was observed by hematoxylin-eosin staining(HE)and Masson staining.The collagen content of lung tissue was detected by hydroxyproline(HYP)method.The expression of Smad7 protein in lung tissue was detected by WB.[Results]The results of pathological staining showed that compared with the control group,lung inflammatory cell infiltration,alveolar wall thickening,and collagen increase were observed after 4 weeks of dustin

关 键 词：石英粉尘 外泌体 miRNA-21-5p SMAD7 肺纤维化 

分 类 号：R11[医药卫生—公共卫生与预防医学]