基于NF-κB信号通路的高通量药物筛选细胞炎症模型的建立

Establishment of a High-throughput Drug Screening Cell Inflammatory Model Based on NF-κB Signaling Pathway

目的建立均相时间分辨荧光(homogeneous time-resolved fluorescence,HTRF)测定炎症细胞模型中NF-κB的方法,为高通量筛选呼吸道炎症药物提供实验依据。方法采用HTRF检测细胞中Total-NF-κB及Phospho-NF-κB比率来确定NF-κB磷酸化程度。观察不同细胞密度(每孔5,25,50,100,150 k)、不同刺激因子(TNF-α、LPS)、不同作用时间(10,30,60 min)等条件对支气管平滑肌细胞(BSMC)、支气管上皮细胞(HBE) NF-κB磷酸化的影响,建立炎症细胞模型及HTRF测定NF-κB的方法。结果采用HBE细胞,细胞密度为每孔10 k,刺激因子TNF-α(10 ng·mL^(-1))刺激细胞,刺激10 min建立炎症细胞模型,可使细胞中NF-κB磷酸化水平达到52.6%,与正常对照组(14.2%)相比,有明显升高。结论成功建立了快速、特异性高、操作简便的HTRF测定细胞炎症模型中NF-κB磷酸化程度的方法,为气道炎症药物筛选研究提供了依据。

OBJECTIVE To establish the homogeneous time-resolved fluorescence(HTRF) detection method of NF-κB on cells inflammatory model, and provide the experiment evidence for high throughput screening of respiratory inflammatory drugs. METHODS Confirmed the extent of phosphorylation of NF-κB through testing the ratio of Total-NF-κB and Phospho-NF-κB by HTRF. Observed the extent of phosphorylation of NF-κB in the different conditions of cell density(10, 25, 50, 100, 150 k per well) of bronchial smooth muscle cells(BSMC) and bronchial epithelial cells(HBE), stimulus factors(TNF-α, LPS), and action time(10, 30, 60 min), built the cell inflammatory model and the detection method of NF-κB by HTRF. RESULTS HBE cells were used, the cell density was 10 k per well, the stimulating factor TNF-α(10 ng·mL-1) stimulated the cells, and the inflammatory cell model was established by stimulation for 10 min, which could increase the phosphorylation level of NF-κB in the cells to 52.6%. Compared with the normal control group(14.2%), there was a significant increase. CONCLUSION A rapid, specific and simple HTRF method for determining the level of NF-κB phosphorylation in the inflammatory model of cells was established, which provide a basis for the screening of airway inflammatory drugs.