Limitations of the use of MTT assay for screening in drug discovery  

MTT方法在药物筛选研究中的应用限制(英文)

作　　者：韩梅[1,2] 李金凤[2] 谭祺[2] 孙媛媛[2] 王永炎[1] 

机构地区：[1]北京师范大学资源学院,北京100875 [2]北京师范大学化学学院放射性药物化学教育部重点实验室,北京100875

出　　处：《Journal of Chinese Pharmaceutical Sciences》2010年第3期195-200,共6页中国药学（英文版）

基　　金：National Natural Science Foundation of China (Grant No.30672491);Beijing New Medical Discipline Based Group(Grant No.XK 100270569)

摘　　要：The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay is a routine cell viability assay for cell proliferation and cytotoxicity, which is widely used in many fields, especially in screening for drug discovery. However, this assay exhibits limitations in the presence of particular compounds and under certain assay conditions, which may yield false screening results. For example, polyphenols that are extracted from natural sources can react with MTT in the absence of living cells and thus interfere with the screening results. We measured the absorbance of 15 polyphenols extracted from green tea and showed that the phenolic hydroxyl groups in the polyphenols are responsible for the reduction of MTT to formazan. When three or more phenolic hydroxyl groups were present on a conjugated polyphenol, a significantly increased MTT reduction was observed. Moreover, the type of medium also had an effect on the absorbance value, in the following order： ct-MEM ＋ 10% FBS〉 a-MEM〉DMEM/F12〉PBS. The absorbance of the MTT assay recorded at 570 nm is more sensitive than that measured at 595 nm. These results will improve the cell-based assay of polyphenols and clarify the limitations of the MTT assay as a method of screening in drug discovery.3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法是一种常规的检测细胞活性和药物毒性的方法,应用范围十分广泛,是在细胞水平的药物筛选研究中重要的检测手段之一。然而,这种检测方法由于化合物或测试条件的原因,可能给出不真实的筛选结果。例如,从天然植物中所提取多酚类化合物可以直接与MTT反应,即使在没有细胞的情况下,随着其浓度的增加,MTT的吸光度值也随之增加,因此不能真实地反映筛选结果。我们从绿茶中提取的15个多酚类化合物进行测定,表明MTT与多酚类化合物反应能力的大小与多酚类酚羟基的还原性有关。当三个或更多的酚羟基形成其轭的多酚时,其还原能力显著增加。同时常用的细胞培养基也能影响MTT的吸光度值,其影响顺序的大小为:α-MEM培养基+10%胎牛血清(FBS)>α-MEM培养基>DMEM/F12>PBS。而且我们还发现MTT法在570 nm处的吸收测定比在595 nm处更敏感。这些发现不仪有助于多酚类化合物的细胞水平活性研究,还将有助于MTT方法在药物筛选研究中的正确应用。

关 键 词：MTTassay Po（yphenols Drug discovery SCREENING 

分 类 号：R965[医药卫生—药理学]