基于高内涵细胞成像系统研究黄芩苷对人肺癌A549细胞上皮间质转化模型的影响  

Using High-Content Screening to Study the Effect of Baicalin on the Epithelial-Mesenchymal Transition Model of Human Lung Cancer A549 Cells

作　　者：徐梦真 许艳 李媛 陈海红[1] 王栋 朱庆均 XU Mengzhen;XU Yan;Li Yuan;CHEN Haihong;WANG Dong;ZHU Qingjun(College of Pharmaceutical Science,Shandong University of Traditional Chinese Medicine,Jinan 250355 Shandong,China;College of Traditional Chinese Medince,Shandong University of Traditional Chinese Medicine,Jinan 250355 Shandong,China;Laboratory Management Office,College of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250355 Shandong,China;The Ministry of Education Antiviral Synergistic Innovation Center of Traditional Chinese Medicine in Universities of Shandong,Jinan 250355 Shandong,China;Innovative Institute of Chinese Medicine and Pharmacy,Shandong University of Traditional Chinese Medicine,Jinan 250355 Shandong,China)

机构地区：[1]山东中医药大学药学院,山东济南250355 [2]山东中医药大学中医学院,山东济南250355 [3]山东中医药大学中医学院实验室管理处,山东济南250355 [4]教育部中医药经典理论研究重点实验室,山东济南250355 [5]山东中医药大学中医药创新研究院,山东济南250355

出　　处：《中药新药与临床药理》2021年第11期1648-1656,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：山东省自然科学基金项目(ZR2020MH384);山东省中医药科技发展计划项目(2019-0040);山东中医药大学大型仪器设备功能开发专项(2018yq17)。

摘　　要：目的基于高内涵细胞成像系统(HCS)探讨黄芩苷(Baicalin)对转化生长因子β1(TGF-β1)诱导的人肺癌A549细胞上皮间质转化(EMT)的影响。方法体外培养人肺癌A549细胞,用TGF-β1诱导A549细胞发生上皮间质转化,设立空白对照组(TGF-β1 0 ng·mL^(-1))、TGF-β1模型组(TGF-β1 5 ng·mL^(-1))和黄芩苷干预组(TGF-β1 5 ng·mL^(-1)+黄芩苷5、10、20μmol·L^(-1))。采用MTT法检测黄芩苷对A549细胞活性的影响;以TGF-β1诱导人肺癌A549细胞发生上皮间质转化,并加入不同浓度黄芩苷干预,采用HCS分析细胞肌动蛋白应激纤维(F-actin)纹理强度、细胞面积、细胞圆度和细胞长度等形态学变化,以及细胞黏附能力和运动迁移能力变化;RT-qPCR法检测上皮间质转化相关标志基因Collagen I、FN1、Vimentin和E-cadherin的相对表达量。结果黄芩苷干预A549细胞48 h后半数抑制浓度(IC50)为22.59μmol·L^(-1)。HCS分析结果显示,与空白对照组比较,TGF-β1模型组细胞形态呈间质细胞样(纺锤形)变化,F-actin纹理强度增强(P<0.01),细胞长度和细胞面积增大(P<0.01),细胞圆度减小(P<0.01),黏附率降低(P<0.01),迁移率和迁移速度升高(P<0.01);与TGF-β1模型组比较,黄芩苷干预组细胞形态呈上皮细胞样(不规则多边形)变化,F-actin纹理强度减弱(P<0.05,P<0.01),细胞长度和细胞面积减小(P<0.01),细胞圆度增大(P<0.01),黏附率升高(P<0.01),迁移率和迁移速度降低(P<0.05,P<0.01)。RT-qPCR结果表明,与空白对照组比较,TGF-β1模型组CollagenⅠ、FN1和Vimentin的mRNA相对表达量升高(P<0.01),E-cadherin的mRNA相对表达量降低(P<0.01);与TGF-β1模型组比较,黄芩苷干预组Collagen I、FN1和Vimentin的mRNA相对表达量降低(P<0.05,P<0.01),E-cadherin的mRNA相对表达量升高(P<0.05,P<0.01)。结论黄芩苷能够有效阻止TGF-β1诱导的人肺癌A549细胞上皮间质转化进程,提示HCS是一种多参数系统分析细胞上皮间质转化的有效工具。Objective To investigate the effect of baicalin on transforming growth factor-β1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)of human lung cancer A549 cells by using high-content screening(HCS).Methods Human lung cancer A549 cells were cultured in vitro,and A549 cells were induced to undergo epithelialmesenchymal transition with TGF-β1.A blank control group(TGF-β1 0 ng·mL^(-1)),a TGF-β1 model group(TGF-β15 ng·mL^(-1))and baicalin intervention group(TGF-β1 5 ng·mL^(-1)+ baicalin 5、10、20 μmol·L^(-1))were established.The MTT method was used to detect the effect of baicalin on the activity of A549 cells.TGF-β1 induced epithelial to mesenchymal transition in A549 cells,and different concentrations of baicalin were added to intervene.HCS was used to analyze morphological changes of cellular actin stress fibers(F-actin),such as texture intensity,cell area,cell roundness and cell length,as well as changes in cell adhesion,movement and migration.RT-qPCR was utilized to measure relative expression of the relative marker genes including Collagen Ⅰ,FN1,Vimentin and Ecadherin for epithelial-mesenchymal transition.Results The half inhibitory concentration(IC50)of baicalin for A549 cells after 48-hour intervention was 22.59 μmol·L^(-1).The results of HCS analysis showed that compared with the blank control group,the cell morphology of the TGF-β1 model group was found mesenchymal cell-like(spindleshaped)changes,texture strength of F-actin increased(P<0.01),cell length and cell area increased(P<0.01),cell roundness decreased(P<0.01),adhesion rate decreased(P<0.01),migration rate and migration speed increased(P<0.01).Compared with the TGF-β1 model group,the cell morphology of the baicalin intervention group was observed epithelial cell-like(irregular polygonal) changes,and the texture intensity of F-actin was weakened(P<0.05,P<0.01).Cell length and cell area decreased(P<0.01),cell roundness increased(P<0.01),adhesion rate increased(P<0.01),migration rate and migration speed decreased(P<0.05,P<0.01)

关 键 词：黄芩苷 高内涵细胞成像系统(HCS) 人肺癌A549细胞 上皮间质转化(EMT)模型 特发性肺纤维化 形态学 细胞迁移 

分 类 号：R285.5[医药卫生—中药学]