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抗结核药物细胞筛选模型的建立与应用研究
Establishment and Application of a cell model for activity screening of new antitubercular drugs
【Author】 Beijing Tuberculosis & Thoracic Tumor Research Institute,Beijing 101149,China Zheng Meiqin Lu Yu Wang Bin Fu Lei
【机构】 北京市结核病胸部肿瘤研究所药物学研究室;
【摘要】 目的建立基于Alamarblue指示剂比色法检测的结核分枝杆菌感染巨噬细胞模型,并应用于氯苯吩嗪衍生物的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/细胞比例感染小鼠巨噬细胞J774A.1不同时间,阳性药异烟肼、利福平、乙胺丁醇与感染细胞作用不同时间,活菌计数测定细胞内菌量,考察最适感染复数(MOI)、感染时间与药物作用时间。以Alamarblue为指示剂,采用比色法测定一些常用抗结核药对感染细胞内菌量的影响,并与活菌计数测定结果进行比较,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。结果感染复数为1:1时,巨噬细胞吞噬菌量为(5.08±0.29)lgCFU/ml;随着MOI增加到10:1,细胞吞噬菌量也明显增加到(6.17±0.13)lgCFU/ml,差异有统计学意义(F=46.454,P<0.001);MOI继续增加到100:1时,细胞吞噬菌量增加到(6.56±0.12)lgCFU/ml,但与MOI为10:1相比,差异无统计学意义(P=0.052),最适宜的感染复数为10:1。巨噬细胞感染结核分枝杆菌2h时,吞噬菌量为(5.81±0.04)lgCFU/ml,感染时间延长到4h时,吞噬菌量也明显增加到(6.07±0.04)lgCFU/ml,差异有统计学意义(F=25.576,P=0.001);但感染时间进一步延长到6h时,吞噬菌量为(6.08±0.07)lgCFU/ml,与感染4h相比,差异无统计学意义(P=0.77),结核分枝杆菌对细胞的感染时间以4h为宜。异烟肼、利福平、乙胺丁醇时效与量效关系考察结果表明药物与感染细胞作用时间以3d为宜。比色法与活菌计数测定常用抗结核药物细胞内抗菌活性的结果相符。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Alamar blue比色法巨噬细胞筛选模型,可对药物细胞内抗结核活性作出快速、客观的评价。
【Abstract】 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 intracellular antitubercular activities of drugs within a short period of time.
- 【会议录名称】 中华医学会结核病学分会2010年学术年会论文汇编
- 【会议名称】中华医学会结核病学分会2010年学术年会
- 【会议时间】2010-09-15
- 【会议地点】中国上海
- 【分类号】R52
- 【主办单位】中华医学会结核病学分会