氨磷汀对噻唑蓝实验结果的影响

Impact of Amifostine on the Result of MTT Assay

目的研究氨磷汀对噻唑蓝(MTT)实验结果的影响。方法实验共分4组:调零组(培养基)、对照组(培养基和细胞)、氨磷汀组(培养基和氨磷汀)、氨磷汀+细胞组(培养基、细胞和氨磷汀),如常规实验加入MTT,立即观察颜色改变及酶联免疫检测仪490 nm处测吸光度(A)值,孵育4 h后再次检测各组A值。结果①调零组、对照组加MTT后均无颜色改变,A值一直在很低水平(0.087±0.021和0.093±0.018),孵育4 h仍无呈色反应,A值也无明显变化(0.076±0.015和0.103±0.022,P>0.05);②氨磷汀组、氨磷汀+细胞组加MTT后均立即呈现蓝紫色,0.5 mmol.L-1氨磷汀时A值分别为0.215±0.013和0.274±0.013,较调零组、对照组均明显升高(P<0.01),且随着氨磷汀浓度增高A值也明显升高(P<0.01);孵育4 h后氨磷汀组A值无继续升高(0.247±0.031,P>0.05),而氨磷汀+细胞组A值仍有升高(0.381±0.022,P<0.05)。结论氨磷汀本身及代谢产物可能与MTT直接反应并呈色,改变MTT实验中的A值,干扰MTT实验结果。

Objective To investigate the impact of amifostine （AMI） on the result of MTT assay. Methods Four groups were arranged as： reset group（ medium）, control group（ medium ＋ cell） .AMI group（ AMI ＋ medium） and AMI ＋ Cell group（ AMI ＋ medium ＋ cell）. At the point of MTT adding and 4 h later, the color of cell culture was observed and the A was detected by enzyme-linked immune assay. Results The results showed that the color of cuhure was unchanged in both reset group and control group, and the of which were low after adding MTT（0.087±0.021 and 0.093±0.018）. Even incubated for 4 hours, the color and A still didn＇t change （0. 076 ± 0. 015 and 0.103 ± 0.022, P 〉 0.05 ） ; the color changed into amethyst in both AMI group and AMI ＋ Cell group, and the A were 0. 215 ＋ 0. 013 and 0. 274 ± 0. 013 after adding MTT with AMI at 0.5 mmol · L^-1, which were significantly higher than those in the reset and control group in a dose-dependent way of AMI. After incubating for d hours, the A ceased increasing in the AM1 group （0. 247 ± 0.031 , P 〉 0.05 ）, but continued to elevate （0.381 ± 0.022, P 〈 0.05 ） in the AMI ＋ Cell group. Conclusion It is concluded that AMI and its metabolites may directly react with MTF and change the color and value,impacting the result of MTT assay.