摘要
气道黏液高分泌是慢性气道炎性疾病的重要临床特征,并且是导致呼吸系统重症发病率升高甚至死亡的关键诱因。目前气道炎症相关毒理学研究主要使用动物模型或二维细胞模型,前者存在效率低的问题,后者存在黏液分泌功能缺失的局限性。本研究采用人支气管上皮细胞(16 human bronchial epithelial cell line,16HBE),使用悬浮培养法将细胞接种到U形底96孔超低吸附板,48 h后形成形状规则的三维球体,直径为(272.7±16.9)μm。使用该三维模型测试7种参考化学品的毒性效应。采用ATP法测试细胞毒性,7种化学品的EC_(50)值为8.2μmol·L^(-1)~668 mmol·L^(-1),与其急性吸入毒性分级相似,且高浓度暴露组细胞球出现解离。进一步在不产生细胞毒性的暴露浓度下原位检测黏蛋白及紧密连接蛋白的表达变化,百草枯、异佛尔酮二异氰酸酯、重铬酸钾、戊二醛、氯化镉使MUC5AC表达升高至对照组的1.24倍~1.34倍。除百草枯外,其余4种化学品使Occludin表达下降16%~33%。乙醇和丙酮没有引起2种蛋白的显著性变化。此外,基于比较毒理基因组学数据库进行慢性气道炎性疾病风险分析,百草枯和肺纤维化、哮喘、炎症相关,异佛尔酮二异氰酸酯诱导气道高反应,氯化镉、重铬酸钾、戊二醛诱导炎症,推理分数为4.37~53.79,乙醇和丙酮无慢性炎性呼吸系统疾病风险,与体外测试结果一致。5种有慢性炎性疾病风险的化学品和呼吸疾病相关差异基因共154个,富集的生物学过程主要包括PI3K、TNF、IL-17、HF-1、EGFR通路等。本研究所构建的人源气道上皮细胞三维模型,可用于检测化学品的黏液高分泌诱导效应,具有成本低、可原位检测及高通量的技术优势,需进一步进行方法验证和机制研究,为开发化学品呼吸毒性筛查新方法提供科学依据。
Airway mucus hypersecretion is an important clinical characteristic of chronic airway inflammatory diseases and a crucial cause of the increased incidence and even death of severe respiratory disorders.Currently,toxicological studies on airway inflammation mainly utilize animal models or two-dimensional cell models.The former has the problem of low efficiency,while the latter has the limitation of losing the mucus secretion function.In this research,16 human bronchial epithelial cell line(16HBE)was inoculated into the U-shaped bottom 96-well ultra-low adsorption plate by the suspension culture method.A three-dimensional sphere with a regular shape was formed 48 hours later,with a diameter of(272.7±16.9)μm.The three-dimensional model was employed to test the toxic effects of 7 reference chemicals.ATP assay was adopted to test cytotoxicity.The EC_(50) values of the 7 chemicals ranged from 8.2μmol·L^(-1)to 668 mmol·L^(-1),which was similar to the classification of acute inhalation toxicity.Furthermore,dissociation occurred in the cell spheres of the high-concentration exposure group.Cell spheres were dissociated in the high exposure concentration group.The expression of mucin and compact junction protein was further detected in situ under non-cytotoxic exposure concentration.The expression of MUC5AC was increased to 1.24-1.34 times that of the control group by paraquat,isophorone diisocyanate,potassium dichromate,glutaraldehyde and cadmium chloride.With the exception of paraquat,the remaining four chemicals decreased O ccludin expression by 16%to 33%.Ethanol and acetone did not cause significant alterations in the two proteins.Additionally,the risk analysis of chronic airway inflammatory diseases was performed based on the comparative toxicological genomic database.Moreover,the risk analysis of chronic airway inflammatory diseases was conducted based on the comparative toxicological genomic database.Paraquat was associated with pulmonary fibrosis,asthma and inflammation;isophorone diisocyanate induced airway hypersensitivity;cadmium chloride,potassium dichromate and glutaraldehyde induced inflammation with reasoning scores ranging from 4.37 to 53.79;ethanol and a cetone had no risk of chronic inflammatory respiratory diseases.It is in line with the outcomes of the in vitro s tudy.There were 154 distinct genes associated with 5 chemicals and respiratory diseases related to the risk of chronic i nflammatory diseases,and the enriched biological processes mainly included PI3K,TNF,IL-17,HF-1,EGFR pathway,etc.The three-dimensional model of human airway epithelial cells constructed in this study can be utilized to detect the mucus hypersecretation-induced effect of chemicals.It possesses the technical advantages of low cost,in-situ detection and high throughput.Further validation of the method and research on the mechanism are required to provide a scientific basis for the development of new methods for screening the respiratory toxicity of chemicals.
作者
朱思瑞
牛雨欣
郎玥明
张博
陈聪
邢立国
刘薇
ZHU Sirui;NIU Yuxin;LANG Yueming;ZHANG Bo;CHEN Cong;XING Liguo;L IU Wei(Key Laboratory of Industrial Ecology and Environmental Engineering,School of Environmental Science and Technology,Dalian University of Technology,Dalian 116024,China;Shenyang Research Institute of Chemical Industry of SINOCHEM Group,Shenyang 110027,China)
出处
《生态毒理学报》
北大核心
2025年第4期15-22,共8页
Asian Journal of Ecotoxicology
基金
国家自然科学基金(22376020)
国家重点研发计划项目课题(2023YFC39005201)
辽宁省科学技术计划项目揭榜挂帅科技攻关专项(2023JH1/10400031)
第17届Mandom国际动物实验替代研究基金。
关键词
慢性气道炎性疾病
三维模型
黏液分泌
屏障功能
chronic airway inflammatory disease
three-dimensional model
mucus secretion
barrier function
作者简介
第一作者:朱思瑞(2000-),男,硕士研究生,研究方向为环境毒理学,E-mail:ihaveadream123@mail.dlut.edu.cn;通信作者:刘薇,E-mail:liu_wei@dlut.edu.cn。
