Ibach B, Appel K, Gebicke-Haerter P, Meyer R P, Friedberg T, Knoth R, Volk B
Department of Neuropathology, Neurocenter, University of Freiburg, Germany.
J Neurosci Res. 1998 Nov 1;54(3):402-11. doi: 10.1002/(SICI)1097-4547(19981101)54:3<402::AID-JNR11>3.0.CO;2-K.
Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti-epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)-labeled cRNA probe demonstrated that 30-40% of the astrocytes strongly expressed a CYP2B mRNA-specific signal within the first week of cultivation. With increasing age (> 14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/polymerase chain reaction (RT/PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression.
对大脑中细胞色素P450 2B（CYP2B）的研究主要集中在蛋白质特性和区域分布上。由于密切相关的CYP2B1和2B2亚型之间序列同源性高，且相应mRNA含量低，因此很少有人致力于分析这些P450基因在特定细胞类型中的表达、调控和诱导性。在本研究中，我们研究了抗癫痫药物苯妥英钠作用下原代大鼠星形胶质细胞培养物中CYP2B mRNA的表达，已知苯妥英钠是肝脏中CYP2B的诱导剂。用地高辛（DIG）标记的cRNA探针进行原位杂交表明，在培养的第一周内，30%-40%的星形胶质细胞强烈表达CYP2B mRNA特异性信号。随着年龄增长（>14天），表达P450 2B mRNA的细胞百分比更高（>90%）。然而，转录活性水平明显低于较年轻的培养物。为了区分2B1和2B2亚型，逆转录/聚合酶链反应（RT/PCR）程序被用于大鼠肝脏mRNA作为对照试验。随后，将该方法应用于培养的星形胶质细胞证实这些脑细胞可能表达CYP2B1 mRNA。在任何检测的年龄中，星形胶质细胞培养物中均未检测到CYP2B mRNA。苯妥英钠导致CYP2B1 mRNA下调，这与该药物对肝脏CYP2B1和2B2水平的诱导作用形成对比。苯妥英钠作用于星形胶质细胞4小时后，根本检测不到扩增产物。苯妥英钠既不诱导CYP2B1也不诱导CYP2B2表达。
