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

关键词:  均相时间分辨荧光  细胞炎症模型  NF-κB

Key words:  homogeneous time-resolved fluorescence  cell inflammatory model  NF-κB

摘要:

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。

Abstract:

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.