应用Alamarblue检测细胞内药物抗结核分枝杆菌活性的研究

Study of Alamar Blue Assay for Determining the Intracellular Activity of New Antitubercular Drugs

目的建立基于Alamarblue检测的结核分枝杆菌感染巨噬细胞模型，应用于氯苯吩嗪衍生物细胞内抗结核活性的测定，为新药筛选提供参数和依据。方法以不同菌量／细胞比例感染巨噬细胞不同时间，阳性药INH、RFP、EMB与感染细胞作用不同时间，CFU计数测定细胞内菌量，考察最适感染复数、感染时间与药物作用时间。以Alamarblue比色法测定一些常用抗结核药对感染细胞内菌量的影响，并与CFU计数测定结果比较，考察比色法的准确性与特异性，并以比色法测定氯苯吩嗪衍生物的细胞内抗结核活性，探讨其在新药筛选中的应用。结果最适感染复数为10：1，感染4h为宜，药物作用时问为3d。比色法与CFU计数测定药物细胞内抗菌活性的结果一致。所测的吩嗪类化合物显示出良好的细胞内抗菌活性。结论建立了Alamarblue比色法巨噬细胞筛选模型，可对细胞内药物抗结核活性作出快速、客观的评价。

Objective To establish an Alamar blue colorimetric macrophage model and apply it to determine the intracellular antitubercular activities of clofazimine derivatives. Methods The optimal MOI, phagocytosed time and drug exposured time were selected based on the bacteria amount in treated macrophages, which was determined by CFU count. The activities of some commonly used antibiotics were investigated within infected macrophages by the colorimetric method and compared with the results obtained from CFU count. The intracellular activities of clofazimine derivatives were determined by the colorimetric method. Results The optimal MOI, phagocytosed time and drug exposured time were 10：1, 4h and 3d respectively. The intracellular activities of the commonly used antibiotics obtained from the colorimetric method were approximate to that from CFU count. The tested clofazimine derivatives showed good intracellular antitubercular activities. Conclusion An Alamar blue colorimetric macrophage model was established and enabled to objectively evaluate the iutracellular antitubercular activities of drugs within a short period of time.