二甲亚砜对细胞色素P450酶3A4和2C9的影响

Effects of Dimethyl Sulfoxide on Cytochrome P450 3A4 and 2C9

目的研究0.01%、0.05%、0.1%二甲亚砜(DMSO)对细胞色素P450(CYP450)酶系中3A4、2C9两个亚型基因及蛋白表达水平的影响。方法 Chang肝脏细胞经处理后,分为空白对照组(仅含培养液)、DMSO组(分别采用0.01%、0.05%、0.1%DMSO处理)、睾酮组(分别采用1、10、100μmol/L睾酮处理)、利福平组(分别采用1、10、100μmol/L利福平处理)、DMSO+睾酮组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L睾酮处理)、DMSO+利福平组(分别采用0.01%、0.05%、0.1%DMSO+10μmol/L利福平处理)。利用逆转录定量PCR的方法,测定CYP3A4、CYP2C9基因水平的表达。利用蛋白免疫印迹法,测定CYP3A4、CYP2C9蛋白水平的表达。结果 0.1%DMSO组CYP3A4 m RNA高于空白对照组(P<0.01),且0.1%DMSO组CYP3A4蛋白的表达也高于空白对照组。0.01%、0.05%DMSO组CYP3A4 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO组CYP3A4蛋白的表达高于空白对照组。0.01%、0.05%、0.1%DMSO组CYP2C9 m RNA表达与空白对照组比较差异无统计学意义(P>0.05),0.01%、0.05%、0.1%DMSO组CYP2C9蛋白表达与空白对照组比较差异无统计学意义(P>0.05)。0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4 m RNA的表达与空白对照组比较差异无统计学意义(P>0.05),但0.01%、0.05%DMSO+10μmol/L睾酮组CYP3A4蛋白的表达高于睾酮组。0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9 m RNA的表达与利福平组比较差异无统计学意义(P>0.05),但0.01%、0.05%、0.1%DMSO+10μmol/L利福平组CYP2C9蛋白的表达高于利福平组。结论0.01%、0.05%、0.1%三个浓度的DMSO在体外肝细胞实验中对CYP3A4的表达有一定的影响,而对CYP2C9的表达影响不明显,当DMSO作为药物溶剂的时候,可能会影响药物单用时的实验结果。

Objective To investigate effects of 0.01%, 0.05%, 0.1% dimethyl sulfoxide（DMSO） on expression of CYP3A4 and CYP2C9 m RNA and protein. Methods Chang liver cells were divided into control group（dealed with RPMI-1640 singly）, DMSO groups（dealed with 0.01%, 0.05%, 0.1% DMSO）, testosterone groups（dealed with 1, 10, 100 μmol/L testosterone）, rifampicin groups（dealed with 1, 10, 100 μmol/L Rifampicin）, DMSO＋testosterone groups（0.01%, 0.05%,0.1% DMSO＋10 μmol/L testosterone） and DMSO＋rifampicin groups（dealed with 0.01%, 0.05%, 0.1% DMSO＋10 μmol/L Rifampicin）. The expression of CYP3A4 and CYP2C9 m RNA were detected by RT-q PCR, and the expression of CYP3A4 and CYP2C9 protein were detected by Western blot. Results Compared with control group, the expression of CYP3A4 m RNA and protein was increased by 0.1% DMSO（P〈0.01）; the expression of CYP3A4 protein could be induced by 0.01% and 0.05% DMSO, but no statistically significant difference on CYP3A4 m RNA（P〈0.05）. Compared with control group, there was no statistically significant difference on the expression of CYP2C9 m RNA and protein in DMSO groups（P〈0.05）. Compared with control group, there was no statistically significant difference on the expression of CYP3A4 m RNA in DMSO＋testosterone groups（P〈0.05）. Compared with testosterone group, the expression of CYP3A4 protein was induced in 0.01%, 0.05%, 0.1%DMSO ＋ testosterone group. Compared with rifampicin group, the expression of CYP2C9 protein was increased in 0.01%, 0.05%, 0.1% DMSO＋Rifampicin group, but no statistically significant difference on CYP2C9 m RNA（P〈0.05）. Conclusion The efficiency of 0.01%, 0.05% and 0.1% DMSO is greater on CYP3A4 than CYP2C9 in vitro test. 0.01%, 0.05%, 0.1% DMSO can influence expression of CYP3A4, buthave poor effect on expression of CYP2C9 in vitro.DMSO as solvent can influence results of experiment.