关键词: 抗结核药, 药物评价, 临床前, 毒性试验, 四唑鎓盐类

Abstract: ObjectiveTo establish MTT-based assay for rapid cytotoxicity screening of new antituberculosis drug and determine the IC₅₀ of clofazimine derivatives. MethodsThe different concentration of Vero and HepG2 cells were prepared with culture medium and seeded into a 96-well microplate.The plate was incubated for 2h to 4h, then 10μl of MTT was added into each well. The plate was reincubated for 4h, and the absorbance was determined. The optimal wavelength was selected based on the optical density values measured at 405nm, 450nm, 492nm and 630nm. Simultaneously, the appropriated cell concentration ranges were characterized according to the curves of cell number vs OD. The cells were seeded into 96-well plates at the appropriated cell concentration and exposed to threefold serial dilutions of the compounds solutions for 48h.The optical density was read at 492nm after incubating with MTT for 4h. The regression curves were estimated from the data with the software origin 7.0. ResultsThe optimal wavelength for MTT was 492nm. The concentrations of cells in 2.5×10⁴～4×10⁵ cells/ml were appropriate for MTT. The IC₅₀s of the 131 compounds tested to Vero cell were approximate to HepG2 cell. The IC₅₀s of 10 compounds were higher than the maximum testing concentration, it suggests that the cytotoxicity was rather low. ConclusionsThe evaluation of Vero cell proliferation effect by MTT-based assay can provide an economical and rapid primary cytotoxicity screening of new antituberculosis drug.

Key words: Antitubercular agents, Drug evaluation,preclinical, Toxicity tests, Tetrazolium salts