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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。
The clinical drug-drug interaction caused by the induction of CYP3A can reduce in vivo exposure and pharmacological activity of CYP3A enzyme substrate drugs. Increased CYP3A mRNA levels are commonly used to assess the induction of CYP3A enzymes by test compounds. This experiment established and verified an absolute quantitative real-time PCR method for the determination of rat liver CYP3A1 and CYP3A2 mRNA expression levels. The designed primers for CYP3A1, CYP3A2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase, housekeeping genes) have high specificity. CYP3A1, CYP3A2 and GAPDH in the concentration range of 1-1 × 10 ~ 6 attomol / μL, the cycle threshold and logarithmic concentration showed a good linear relationship, and has good experimental intra-group and inter-group repeatability. The method was successfully applied to study the changes of the induction of CYP3A1 and CYP3A2 mRNA in the liver of rats after intraperitoneal injection of dexamethasone 100 mg / kg. The basal levels of CYP3A1 and CYP3A2 mRNA in total RNA were 37.78 and 252.31 attomol / μg, respectively. Subsequent CYP3A1 and CYP3A2 mRNA levels gradually increased and reached the maximum induction effect at 24 hours and 42 hours, respectively, 19 times and 8 times the basal level. Finally, CYP3A1 and CYP3A2 mRNA returned to their respective basal levels 60 hours after dexamethasone administration.