Pérez Gabriela, Tabares Beatriz, Jover Ramiro, Gómez-Lechón Maria-José, Castell José V
Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda de Campanar 21, E-46009, Valencia, Spain.
Toxicol In Vitro. 2003 Oct-Dec;17(5-6):643-9. doi: 10.1016/s0887-2333(03)00109-7.
An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional liver tissue are incubated with the drugs for 50 h. Total RNA is extracted from culture and the cDNA is prepared by reverse transcription using high fidelity reverse transcriptase. Using appropriate primers, selective amplification of each CYP cDNA is achieved and real-time quantified by SYBR-Green fluorescence measurement. The extent of CYP induction obtained with the tested compounds is compared with the induction obtained with CYP model inducers (methylcholantrene, phenobarbital and rifampicin). This technique can be of value, to considerably simplify the identification of drug candidates with potential CYP inducing ability in man.
已开发出一种用于定量测量人肝细胞中主要人类细胞色素P450（CYP）同工型（1A1、1A2、2A6、2B6、2C9, 2C19、2D6、2E1、3A4和3A5）mRNA含量的检测方法。该方法基于将mRNA转化为其相应的cDNA，然后使用合适的引物进行PCR扩增。利用合适的内标和外标，可以估计肝细胞中CYP mRNA含量的变化。该技术已标准化以进行半自动操作。此程序可用于在临床前开发阶段评估新药物的CYP诱导潜力。为此，将从功能性肝组织获得的人肝细胞与药物孵育50小时。从培养物中提取总RNA，并使用高保真逆转录酶通过逆转录制备cDNA。使用合适的引物，实现每个CYP cDNA的选择性扩增，并通过SYBR-绿荧光测量进行实时定量。将测试化合物获得的CYP诱导程度与CYP模型诱导剂（甲基胆蒽、苯巴比妥和利福平）获得的诱导程度进行比较。该技术可能具有重要价值，可大大简化在人体中具有潜在CYP诱导能力的候选药物的鉴定。
