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

doi:10.5246/jcps.2011.06.077

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

李良, 李载权, 李汉青, 毕姗姗, 许娇娇, 李博, 周田彦^*, 卢炜^*

1. 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
2. 北京大学医学部药学院药剂学系, 北京 100191
3. 北京大学医学部基础医学院放射医学教研室, 北京 100191

收稿日期:2011-05-30 修回日期:2011-09-20 出版日期:2011-11-15 发布日期:2011-11-15
通讯作者: 周田彦*, 卢炜*

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

Liang Li, Zai-Quan Li, Han-Qing Li, Shan-Shan Bi, Jiao-Jiao Xu, Bo Li, Tian-Yan Zhou^*, Wei Lu^*

1. State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China
2. Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
3. Department of Radiation Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2011-05-30 Revised:2011-09-20 Online:2011-11-15 Published:2011-11-15
Contact: Tian-Yan Zhou*, Wei Lu*

摘要/Abstract

摘要：

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1-1×10⁶ 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CYP3A2 mRNA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: 实时定量PCR, CYP3A1, CYP3A2, mRNA, 地塞米松, 诱导作用

Abstract:

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 1×10⁶ attomol/μL 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/μg (total RNA) and 252.31 attomol/μg (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.

Key words: Real time PCR, CYP3A1, CYP3A2, mRNA, Dexamethasone, Induction

中图分类号:

R945

Supporting:

Foundation items: National Integrity Innovational Technology Platform of New Drug and Development (Grant No. 2009ZX09301-010).
^*Corresponding author. Tel.: 86-10-82801717

李良, 李载权, 李汉青, 毕姗姗, 许娇娇, 李博, 周田彦^*, 卢炜^*. 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[J]. , DOI: 10.5246/jcps.2011.06.077.

Liang Li, Zai-Quan Li, Han-Qing Li, Shan-Shan Bi, Jiao-Jiao Xu, Bo Li, Tian-Yan Zhou^*, Wei Lu^*. Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay[J]. , DOI: 10.5246/jcps.2011.06.077.

参考文献

[1] Shou, M.; Hayashi, M.; Pan, Y.; Xu, Y.; Morrissey, K.; Xu, L.; Skiles, G.L. Drug Metab. Dispos. 2008, 36, 2355-2370.
[2] Dickins, M. Curr. Top. Med. Chem. 2004, 4, 1745-1766.
[3] Kliewer, S.A.; Moore, J.T.; Wade, L.; Staudinger, J.L.; Watson, M.A.; Jones, S.A.; McKee, D.D.; Oliver, B.B.; Willson, T.M.; Zetterstrom, R.H.; Perlmann, T.; Lehmann, J.M. Cell. 1998, 92, 73-82.
[4] Kuschnaroff, L.M.; Valckx, D.; Goebels, J.; Rutgeerts, O.; Heremans, H.; Froyent, G.; Waer, M. Scand. J. Immunol. 1997, 46, 459-468.
[5] Zhou, N.M.; Matthys, P.; Polacek, C.; Fiten, P.; Sato, A.; Billiau, A.; Froyen, G. Cytokine. 1997, 9, 212-218.
[6] Fox, C.J.; Danska, J.S. J. Immunol. 1997, 158, 2414-2424.
[7] Hanson, M.S.; Cetkovic-Cvrlje, M.; Ramiya, V.K.; Atkinson, M.A.; Maclaren, N.K.; Singh, B.; Elliott, J.F.; Serreze, D.V.; Leiter, E.H. J. Immunol. 1996, 157, 1279-1287.
[8] Overbergh, L.; Valckx, D.; Waer, M.; Mathieu, C. Cytokine. 1999, 11, 305-312.
[9] Okano, Y.; Hristova, K.R.; Leutenegger, C.M.; Jackson, L.E.; Denison, R.F.; Gebreyesus, B.; Lebauer, D.; Scow, K.M. Appl. Environ. Microbiol. 2004, 70, 1008-1016.
[10] Burczynski, M.E.; McMillian, M.; Parker, J.B.; Bryant, S.; Leone, A.; Grant, E.R.; Thorne, J.M.; Zhong, Z.; Zivin, R.A.; Johnson, M.D. Drug Metab. Dispos. 2001, 29, 1243-1250.
[11] Cui, X.; Thomas, A.; Han, Y.; Palamanda, J.; Montgomery, D.; White, R.E.; Morrison, R.A.; Cheng, K.C. J. Pharmacol. Toxicol. Methods. 2005, 52, 234-243.
[12] Mei, Q.; Richards, K.; Strong-Basalyga, K.; Fauty, S.E.; Taylor, A.; Yamazaki, M.; Prueksaritanont, T.; Lin, J.H.; Hochman, J. J. Pharm. Sci. USA. 2004, 93, 2488-2496.
[13] Martignoni, M.; de Kanter, R.; Grossi, P.; Mahnke, A.; Saturno, G.; Monshouwer, M. Chem. Biol. Interact. 2004, 151, 1-11.
[14] Hutmacher, D.W.; Leong, D.T.; Gupta, A.; Bai, H.F.; Wan, G.Q.; Yoong, L.F.; Too, H.P.; Chew, F.T. Biomaterials. 2007, 28, 203-210.
[15] Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. Biotechnology (N Y). 1993, 11, 1026-1030.
[16] Martin, B.; Jofre, A.; Garriga, M.; Pla, M.; Aymerich, T. Appl. Environ. Microbiol. 2006, 72, 6040-6048.
[17] O'Connell, K.P.; Bucher, J.R.; Anderson, P.E.; Cao, C.J.; Khan, A.S.; Gostomski, M.V.; Valdes, J.J. Appl. Environ. Microbiol. 2006, 72, 478-483.
[18] Rodriguez-Lazaro, D.; Lewis, D.A.; Ocampo-Sosa, A.A.; Fogarty, U.; Makrai, L.; Navas, J.; Scortti, M.; Hernandez, M.; Vazquez-Boland, J.A. Appl. Environ. Microbiol. 2006, 72, 4256-4263.
[19] Mahnke, Z.; Strotkamp, D.; Roos, P.H.; Hanstein, W.G.; Chabot, G.G.; Nef, P. Arch. Biochem. Biophys. 1997, 337, 62-68.
[20] Hoen, P.A.; Commandeur, J.N.; Vermeulen, N.P.; Van Berkel, T.J.; Bijsterbosch, M.K. Biochem. Pharmacol. 2000, 60, 1509-1518.
[21] Matsubara, T.; Kim, H.J.; Miyata, M.; Shimada, M.; Nagata, K.; Yamazoe, Y. J. Pharmacol. Exp. Ther. 2004, 309, 1282-1290.
[22] Huss, J.M.; Wang, S.I.; Kasper, C.B. Arch. Biochem. Biophys. 1999, 372, 321-332.