PharmaCon GmbH, Riglergasse 4/5, 1180 Vienna, Austria.
Neurosci Lett. 2010 Jan 22;469(2):224-8. doi: 10.1016/j.neulet.2009.11.079. Epub 2009 Dec 4.
The blood-brain barrier (BBB) maintains the homeostasis between the central nervous system and the blood circulation. One of the main efflux transporter proteins at the BBB is P-glycoprotein (P-gP) also known as ABCB1 or MDR1. Due to the important role of P-gP for the transport barrier function of the BBB, the presence and functionality of P-gP was investigated in porcine cell line PBMEC/C1-2. Presence of P-gP was confirmed on the protein level by western blotting and immunofluorescence microscopy as well as on the mRNA level by qPCR. Functional assessment was accomplished by an established 96-well uptake assay using Rhodamine 123 and Doxorubicin as P-gP substrates and Verapamil as moderate P-gP inhibitor. In this regard, fluorescence microscopy confirmed a significant higher uptake of Rhodamine 123 into PBMEC/C1-2 cells when preincubated with Verapamil. Finally, knock-down of P-gP by antisense oligonucleotides revealed an increase of Rhodamine 123 uptake indicating decreased P-gP functionality. In summary, the presence and functionality of P-gP in the immortalised cell line PBMEC/C1-2 was proven with several techniques and assays. Thus, this cell line could be used for P-gP studies in the context of BBB relevant issues.
血脑屏障（BBB）维持着中枢神经系统和血液循环之间的内环境稳定。BBB 上的主要外排转运蛋白之一是 P 糖蛋白（P-gP），也称为 ABCB1 或 MDR1。由于 P-gP 对 BBB 的转运屏障功能非常重要，因此在猪细胞系 PBMEC/C1-2 中研究了 P-gP 的存在和功能。通过 Western blot 和免疫荧光显微镜在蛋白质水平上以及通过 qPCR 在 mRNA 水平上证实了 P-gP 的存在。通过使用 Rhodamine 123 和 Doxorubicin 作为 P-gP 底物以及 Verapamil 作为中度 P-gP 抑制剂的已建立的 96 孔摄取测定法完成了功能评估。在这方面，荧光显微镜证实，当用 Verapamil 预孵育时，PBMEC/C1-2 细胞中 Rhodamine 123 的摄取明显更高。最后，通过反义寡核苷酸敲低 P-gP 显示 Rhodamine 123 摄取增加，表明 P-gP 功能降低。总之，通过多种技术和测定法证明了永生化细胞系 PBMEC/C1-2 中 P-gP 的存在和功能。因此，该细胞系可用于与 BBB 相关问题相关的 P-gP 研究。
