Chan Gary N Y, Bendayan Reina
Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2011;686:313-36. doi: 10.1007/978-1-60761-938-3_15.
The blood-brain barrier (BBB) physically and metabolically functions as a neurovascular interface between the brain parenchyma and the systemic circulation, and regulates the permeability of several endogenous substrates and xenobiotics in and out of the central nervous system. Several membrane-associated transport proteins, such as P-glycoprotein (P-gp), multidrug resistance-associated proteins, breast cancer resistance protein, and organic anion transporting polypeptides, have been characterized at the BBB and identified to play a major role in regulating the brain bioavailability of several pharmacological agents. This chapter reviews several well-established techniques for the study of the molecular expression, cellular localization, and functional activity of transport proteins in primary and immortalized cell culture systems of the BBB. In particular, we describe the molecular characterization of P-gp/MDR1 at the transcript level using semiquantitative polymerase chain reaction (PCR), at the protein level using immunoblotting, and at the cellular level using immunofluorescence. In addition, the uptake/efflux and transepithelial flux studies, which characterize P-gp transport activity, are described.
血脑屏障（BBB）在物理和代谢方面作为脑实质与体循环之间的神经血管界面发挥作用，并调节几种内源性底物和外源性物质进出中枢神经系统的通透性。几种与膜相关的转运蛋白，如P-糖蛋白（P-gp）、多药耐药相关蛋白、乳腺癌耐药蛋白和有机阴离子转运多肽，已在血脑屏障中得到表征，并被确定在调节几种药物的脑生物利用度方面起主要作用。本章综述了几种成熟的技术，用于研究血脑屏障原代和永生化细胞培养系统中转运蛋白的分子表达、细胞定位和功能活性。特别是，我们描述了使用半定量聚合酶链反应（PCR）在转录水平、使用免疫印迹在蛋白质水平以及使用免疫荧光在细胞水平对P-gp/MDR1进行分子表征。此外，还描述了表征P-gp转运活性的摄取/外排和跨上皮通量研究。
