肝细胞共培养模型建立及铅、镉、汞、砷混合染毒的联合作用模式分析  

Establishment of liver cells co-culture model and analysis of the combined action mode of lead,cadmium,mercury and arsenic mixed toxicity

作　　者：段爱茹 乔梦芸 鱼涛[1] 陈宵[1] 张意[1] 刘黎[1] 王海华[1] 肖经纬[1] 吕书俊 吴思雨 佟璐瑶 李斌[1] DUAN Ai-ru;QIAO Meng-yun;YU Tao;CHEN Xiao;ZHANG Yi;LIU Li;WANG Hai-hua;XIAO Jing-wei;LV Shu-jun;WU Si-yu;TONG Lu-yao;LI-Bin(National Institute for Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,National Key Laboratory of Trauma and Chemical Poisoning,Beijing 100050,China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,中国疾病预防控制中心创伤与化学中毒全国重点实验室,北京100050

出　　处：《毒理学杂志》2024年第5期329-337,343,共10页Journal of Toxicology

基　　金：十四五国家重点研发项目“职业肿瘤与职业性听力损失的风险评估技术和生物标志物研究”(2022YFC2503203)。

摘　　要：目的探讨实际暴露比例下的铅、镉、汞和砷混合染毒对肝细胞的联合作用模式,并建立模拟人体微环境的肝细胞共培养模型。方法使用ICP-MS测定某铅锌冶炼厂重金属暴露工人血液铅、镉、汞和砷浓度比。采用MTT法检测细胞存活率并筛选共培养细胞模型的重金属混合染毒浓度。使用Transwell共培养小室,建立以HepG2细胞为肝实质细胞、THP-1诱导来源的巨噬细胞和LX-2细胞为肝非实质细胞的共培养模型。结果工人血液铅、镉、汞、砷浓度比约为266:4:5:11。在<IC_(40)时(IC_(10)~IC_(30)),4种重金属对HepG2细胞的联合作用表现为协同作用,随着细胞抑制浓度的升高,CI值增大,协同作用的强度减小;在>IC_(40)(IC_(50)~IC_(90))时,4种重金属对HepG2细胞的联合作用表现为拮抗作用,随着细胞抑制浓度的进一步升高,CI值更加增大,拮抗作用的强度明显增大,上述联合作用模式分析结果在染毒24、48和72h一致。在=IC_(40)时,染毒24h4种重金属对HepG2细胞的联合作用表现为轻度拮抗作用,而染毒48和72h 4种重金属对HepG2细胞的联合作用表现为接近于相加作用。根据共培养模型中3种成分细胞HepG2细胞、THP-1诱导来源的巨噬细胞和LX-2细胞的MTT实验结果及IC_(50),选定后续研究的混合染毒浓度为:铅+镉+汞+砷(66.5+1+1.25+2.75)μmol/L,并以此浓度染毒共培养模型,染毒24h后,共培养模型中HepG2细胞相对存活率低于单独培养HepG2细胞相对存活率(t_(24h)=5.704,P<0.05),染毒24和48h后,共培养模型中HepG2细胞线粒体膜电位高于单独培养HepG2细胞(t_(24h)=3.49,t_(48h)=3.941,P<0.05),共培养模型中HepG2细胞ROS水平低于单独培养HepG2细胞(t_(24h)=1.048,t_(48h)=2.946,P<0.05)。结论铅、镉、汞和砷混合染毒对HepG2细胞的联合作用在不同染毒时间和抑制浓度下表现不同:IC_(10)~IC_(30)表现为协同作用,IC_(50)-IC_(90)表现为拮抗作用,IC_(40)是联合作用模式发生改变的Objective e To explore the combined toxic effects of lead,cadmium,mercury and arsenic under actual exposure proportions on liver cells,and establish a co-culture model of liver cells to simulate the human body micro-environment.Methods sUsing ICP-MS to determine the concentration ratios of lead,cadmium,mercury and arsenic in the blood of heavy metal exposed workers in a lead-zinc smelting plant.Using MTT assay to detect cell survival rate and screen for heavy metal mixed toxicity concentration in co cultured cell models.Using Transwell co-culture chamber,to establish a co-culture model with HepG2 cells as liver parenchymal cells,THP-1-induced macrophages,and LX-2 cells as liver non parenchymal cells was established.Results The ratio of lead,cadmium,mercury and arsenic concentrations in the blood of workers is approximately 266:4:5:11.When the concentration is below IC_(40)(IC_(10)~IC_(30)),the combined effect of the four heavy metals on HepG2 cells shows a synergistic effect.As the cell inhibition concentration increases,the CI value increases,and the strength of the synergistic effect decreases.When above IC_(40)(IC_(50)~IC_(90)),the combined effect of the four heavy metals on HepG2 cells shows an antagonistic effect.With further increases in cellinhibition concentration,the CI value significantly increases,and the strength of the antagonistic effect notably increases.The analysis of the above combined effect patterns is consistent at 24,48,and 72 hours of exposure.At IC_(40),after 24 hours of exposure,the combined effect of the four heavy metals on HepG2 cells shows a mild antagonistic effect,while after 48 and 72 hours of exposure,the combined effect shows an effect close to an additive one.Based on the MTT assay result and ICso of the three components HepG2 cells,THP-1-induced macrophages and LX-2 cells in the co-culture model,a mixed exposure concentration for subsequent studies is selected as follows:lead+cadmium+mercury+arsenic(66.5+1+1.25+2.75)μmol/L.Using this concentration for co-culture,after 24 hours

关 键 词：重金属混合染毒 联合作用 肝细胞共培养模型 

分 类 号：R135.1[医药卫生—劳动卫生] R99[医药卫生—公共卫生与预防医学] R114