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

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).RT-qPCR results showed that,compared with the blank control group,the relative mRNA expression of Collagen Ⅰ,FN1 and Vimentin in the TGF-β1 model group increased(P<0.01),and the relative mRNA expression of E-cadherin decreased(P<0.01).Compared with the TGF-β1 model group,the relative mRNA expression of Collagen Ⅰ,FN1 and Vimentin in the baicalin intervention group decreased(P<0.05,P<0.01),and the relative mRNA expression of E-cadherin increased(P<0.05,P<0.01).Conclusion Baicalin can effectively prevent the TGF-β1 induced epithelial-mesenchymal transition process.The study also suggests that HCS is an effective tool for multi-parameter analysis of cell epithelial-mesenchymal transition.