Pan J, Xiang Q, Ball S
Wyeth-Ayerst Research, Department of Drug Safety and Metabolism, Princeton, New Jersey, USA.
Drug Metab Dispos. 2000 Jun;28(6):709-13.
In this study, we developed a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to study cytochrome P450 (CYP) mRNA regulation by cytokines in mouse liver. The method combines standard RT-PCR with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target template present. We show that this method provides very rapid, sensitive, and reproducible quantification of mouse Cyp mRNA with a wide dynamic range of starting target molecule. We validated the method by using several prototypic CYP inducers. As expected, the mRNA levels of Cyp3a11, Cyp2b10, and Cyp1a2 were induced by a single dose of dexamethasone (100 mg/kg), phenobarbital (80 mg/kg), and 3-methylcholanthrene (80 mg/kg), respectively. The method of real-time RT-PCR was then used to evaluate the effects of interleukin (IL)-6 (100 ng/mouse), IL-1beta (500 ng/mouse), and tumor necrosis factor (TNF)-alpha (2 microgram/mouse) on Cyp mRNA expression in the mouse. Constitutive Cyp2b10 mRNA was reduced to 40% by IL-6 and 15% by IL-1beta, whereas Cyp2d9 mRNA was reduced to 70% by TNF-alpha administration. The level of Cyp1a2 mRNA was suppressed to 67% by IL-6 and 59% by TNF-alpha. Cyp3a11 and Cyp2e1 mRNAs were not affected by any of the cytokines that were studied. We conclude that the real-time RT-PCR method is a powerful new tool to study CYP mRNA expression and regulation. Using this method, we are the first to report that the expression of constitutive Cyp2b10, 1a2, and 2d9 mRNAs was suppressed by proinflammatory cytokines.
在本研究中，我们开发了一种实时定量逆转录-聚合酶链反应（RT-PCR）方法，用于研究细胞因子对小鼠肝脏中细胞色素P450（CYP）mRNA的调控。该方法将标准RT-PCR与一种荧光探针相结合，荧光强度与存在的靶模板量成正比。我们表明，该方法能在很宽的起始靶分子动态范围内，对小鼠Cyp mRNA进行非常快速、灵敏且可重复的定量。我们通过使用几种典型的CYP诱导剂验证了该方法。正如预期的那样，单次给予地塞米松（100 mg/kg）、苯巴比妥（80 mg/kg）和3-甲基胆蒽（80 mg/kg）分别诱导了Cyp3a11、Cyp2b10和Cyp1a2的mRNA水平。然后使用实时RT-PCR方法评估白细胞介素（IL）-6（100 ng/小鼠）、IL-1β（500 ng/小鼠）和肿瘤坏死因子（TNF）-α（2微克/小鼠）对小鼠Cyp mRNA表达的影响。组成型Cyp2b10 mRNA被IL-6降低至40%，被IL-1β降低至15%，而Cyp2d9 mRNA在给予TNF-α后降低至70%。Cyp1a2 mRNA水平被IL-6抑制至67%，被TNF-α抑制至59%。Cyp3a11和Cyp2e1 mRNA不受所研究的任何细胞因子影响。我们得出结论，实时RT-PCR方法是研究CYP mRNA表达和调控的一种强大新工具。使用该方法，我们首次报道促炎细胞因子抑制了组成型Cyp2b10、1a2和2d9 mRNA的表达。
