慢性阻塞性肺疾病相关性气管支气管软化症模型建立及评价  

The model establishment method and evaluation of chronic obstructive pulmonary disease-associated tracheobronchomalacia

作　　者：李雪莲 余薇[2] 周鹏程[3] LI Xue-Lian;YU Wei;ZHOU Peng-Cheng(Emergency Department of Sichuan Second Hospital of Traditional Chinese Medicine,Chengdu 610031,Sichuan,China)

机构地区：[1]四川省第二中医医院急诊科,四川成都610031 [2]成都中医药大学临床医学院 [3]成都中医药大学附属医院呼吸内科

出　　处：《中国老年学杂志》2023年第24期5971-5978,共8页Chinese Journal of Gerontology

基　　金：国家自然科学基金(81904177);成都中医药大学杏林学者科研提升计划(QNXZ2020007);成都中医药大学附属医院百人计划(20-Q07);成都中医药大学附属医院科技发展基金(22ZL03)。

摘　　要：目的建立一种慢性阻塞性肺疾病(COPD)相关性气管支气管软化症模型制备方法,并评价丝裂原活化蛋白激酶(p38MAPK)抑制剂(SB203580)的作用效果。方法通过烟熏联合脂多糖气管滴入法制备大鼠COPD模型,分离COPD大鼠气管支气管软骨细胞、培养及传代,然后加入10 ng/ml白细胞介素(IL)-1β处理筛选的软骨细胞24 h诱导软骨细胞退变,建立COPD相关性气管支气管软化症模型。采用SB203580干预退变软骨细胞,流式凋亡和CCK8法分别检测软骨细胞凋亡、增殖活性;甲苯胺蓝染色和免疫组织化学法观察软骨细胞蛋白聚糖和Ⅱ型胶原含量;Western印迹检测软骨细胞caveolin-1、p38MAPK、IL-1β、基质金属蛋白酶(MMP)-13蛋白表达;荧光定量聚合酶链反应(qPCR)法检测软骨细胞caveolin-1、p38MAPK、IL-1β、MMP-13表达水平。结果COPD大鼠支气管软骨细胞分离鉴定成功,10 ng/ml IL-1β即能诱导构建稳定的COPD相关性气管支气管软化症细胞模型;通过CCK8法筛选确定第4代支气管软骨细胞、5μmol/L SB203580为最佳传代细胞和干预剂量,依上述条件分别处理各组软骨细胞48 h。甲苯胺蓝染色和免疫组织化学分析表明,SB203580处理可以有效提高COPD相关性气管支气管软化症细胞模型软骨细胞的蛋白聚糖和Ⅱ型胶原含量;细胞流式检测结果显示:SB203580可以显著降低细胞凋亡,显著增强细胞增殖活性(P<0.05);Western印迹检测结果表明:SB203580可以有效抑制caveolin-1、p38MAPK、MMP-13、1L-1β蛋白表达;qPCR检测结果表明SB203580可以有效抑制MMP-13、p38MAPK、caveolin-1、IL-1β基因的表达。结论10 ng/ml IL-1β可诱导构建稳定的COPD相关性气管支气管软化症细胞模型;SB203580可提高该模型软骨细胞的蛋白聚糖和Ⅱ型胶原含量及提高细胞活性,同时减少软骨细胞凋亡。其作用可能与SB203580抑制caveolin-1-p38MAPK信号通路中关键信号分子caveolin-1、p38MAPK表达及降低下游效�Objective To establish a model preparation method of chronic obstructive pulmonary disease(COPD)-associated tracheobronchomalacia and to evaluate the effect of mitogen-activated protein kinase(p38MAPK)inhibitor(SB203580).Methods A rat model of COPD was prepared by smoking combined with tracheal lipopolysaccharide injection.The trachea or bronchus chondrocytes from COPD rats were isolated,cultured,passaged,and chondrocytes were treated with 10 ng/ml interleukin(IL)-1βfor 24 h to induce chondrocyte degeneration and to develop a model of COPD-associated tracheobronchomalacia.The degenerative chondrocytes were treated with SB203580.Flow cytometry and CCK8 assays were used to detect apoptosis and proliferative activity,respectively.Toluidine blue staining and immunohistochemistry were used to observe the chondrocyte proteoglycan and typeⅡcollagen content.Western blotting was used to detect the expressions of caveolin-1,p38MAPK,IL-1β,matrix metalloproteinase(MMP)-13 protein in chondrocyte.Quantitative polymerase chain reaction(PCR)was used to detect the expression levels of caveolin-1,p38MAPK,IL-1β,MMP-13 in chondrocyte.Results The isolation and identification of bronchial chondrocytes from COPD rats was successful.10 ng/ml IL-1βcould induce the construction of a stable COPD-associated tracheobronchomalacia chondrocyte model.CCK8 method was used to determine that the fourth-generation bronchial chondrocytes were the best passage cells and 5μmol/L SB203580 was the best intervention dose,and the corresponding groups of chondrocytes were treated for 48 h under the above conditions.Toluidine blue staining and immunohistochemical analysis showed that SB203580 treatment could effectively increase the content of proteoglycan and typeⅡcollagen in chondrocytes of COPD associated tracheobronchomalacia cell model.The result of flow cytometry showed that SB203580 could significantly reduce cell apoptosis and significantly enhance cell proliferation activity(P<0.05).The result of Western blotting showed that SB203580 could

关 键 词：慢性阻塞性肺疾病相关性气管支气管软化症 试验模型 丝裂原活化蛋白激酶(p38MAPK)抑制剂(SB203580) caveolin-1-p38MAPK信号通路 

分 类 号：R332[医药卫生—人体生理学]