Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay  

实时定量PCR法绝对定量检测大鼠肝脏CYP3A1和CYP3A2 mRNA的诱导表达(英文)

作　　者：李良[1] 李载权[2] 李汉青[1] 毕姗姗[1] 许娇娇[1] 李博[2] 周田彦[3,1] 卢炜[3,1] 

机构地区：[1]北京大学医学部药学院药剂学系,北京100191 [2]北京大学医学部基础医学院放射医学教研室,北京100191 [3]北京大学医学部天然药物及仿生药物国家重点实验室,北京100191

出　　处：《Journal of Chinese Pharmaceutical Sciences》2011年第6期597-603,共7页中国药学（英文版）

基　　金：National Integrity Innovational Technology Platform of New Drug and Development(Grant No.2009ZX09301- 010)

摘　　要：Clinical drug-drug interactions（DDIs） induced by CYP3A may reduce the exposure and pharmacological activity of CYP3A substrate.Up-regulation of CYP3A mRNA is often used to evaluate inductive effect of test compounds on CYP3A. A quantitative real time PCR assay was developed and validated for the absolute quantification of CYP3A1 and CYP3A2 mRNA.Specific primers of CYP3A1,CYP3A2 and GAPDH（glyceraldehyde-3-phosphate dehydrogenase,as a house-keeping gene） were well designed.The relationship between threshold cycle（Ct） and logarithm of the concentrations of CYP3A1, CYP3A2 and GAPDH was linear ranged from 1 attomol/μL to 1×10~6 attomol/uL with great inter- and intra-assay reproducibility. This method was successfully applied to investigate the time courses of CYP3A1 and CYP3A2 mRNA induction in rat liver after 100 mg/kg dexamethasone（DEX） administration by intraperitoneal（i.p.） injection.The baseline levels of CYP3A1 and CYP3A2 mRNAs were 37.78 attomol/ug（total RNA） and 252.31 attomol/ug（total RNA）,respectively.CYP3A1 and CYP3A2 mRNA values increased gradually to their peak levels（19- and 8- fold vs.baseline） within 24 h and 42 h,respectively,and then returned to their baseline 60 h after DEX administration.由CYP3A诱导作用引起的临床药物-药物间相互作用能同时降低CYP3A酶底物药物的体内暴露量和药理活性。CYP3A mRNA水平的增高常用于评价受试化合物对CYP3A酶的诱导作用。本实验建立并验证了一种绝对定量的实时PCR方法用于测定大鼠肝中CYP3A1和CYP3A2 mRNA的表达水平。设计的CYP3A1、CYP3A2和GAPDH（甘油醛-3-磷酸脱氢酶,看家基因）引物具有很高的特异性。CYP3A1、CYP3A2和GAPDH在1-1×10~6 attomol/μL浓度范围内,循环阈值和对数浓度呈现良好的线性关系,并且具有良好的实验组内和组间可重复性。该方法成功应用于考察大鼠腹腔注射给予100 mg/kg地塞米松后肝脏中CYP3A1和CYP3A2 mRNA诱导作用随时间的变化规律。总RNA中CYP3A1和CYP3A2mRNA的基础水平分别为37.78和252.31 attomol/μg。随后CYP3A1和CYP3A2 mRNA水平逐渐增加并分别在24小时和42小时达到最大诱导效应,分别为基础水平的19倍和8倍。最终CYP3A1和CYP3A2 mRNA在地塞米松给药60小时后回落到各自的基础水平。

关 键 词：Real time PCR CYP3A1 CYP3A2 MRNA DEXAMETHASONE INDUCTION 

分 类 号：R96[医药卫生—药理学]