Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan, ROC.
Colloids Surf B Biointerfaces. 2011 Oct 1;87(1):139-45. doi: 10.1016/j.colsurfb.2011.05.014. Epub 2011 May 12.
The expressions of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) in the blood-brain barrier (BBB) were regulated by verapamil and probenecid. The two protein interveners inhibited the efflux-pumping capability for permeating calcein-AM in the monolayer of human brain-microvascular endothelial cells (HBMECs). The immunochemical staining revealed that the order in the integrity of tight junction was human astrocyte (HA)-regulated HBMECs>HBMECs cultured with 100% astrocyte-conditioned medium (ACM)>HBMECs cultured with 50% ACM>HBMECs. The viability of HBMECs was higher than 94% when the concentrations of verapamil and probenecid were lower than 50 μM and 1000 μM, respectively. The culture using ACM negligibly affected the activity of P-gp and MRPs on HBMECs after the suppression with verapamil and/or probenecid. However, the double culture of HA-regulated HBMECs promoted the quantity of P-gp and MRPs and reduced the endocytosis of calcein-AM. The inhibitive and endocytotic analysis can unveil the role of HAs in the protein expressions on HBMECs for establishing a reliable BBB model in vitro.
维拉帕米和丙磺舒可调节血脑屏障（BBB）中 P-糖蛋白（P-gp）和多药耐药相关蛋白（MRPs）的表达。这两种蛋白干预剂抑制了单层人脑微血管内皮细胞（HBMEC）中 calcein-AM 的渗透外排泵能力。免疫化学染色显示，紧密连接完整性的顺序为星形胶质细胞（HA）调控的 HBMEC>HBMEC 用 100%星形胶质细胞条件培养基（ACM）培养>HBMEC 用 50%ACM 培养>HBMEC。当维拉帕米和丙磺舒的浓度分别低于 50μM 和 1000μM 时，HBMEC 的活力高于 94%。用 ACM 培养对 HBMEC 中 P-gp 和 MRPs 的活性几乎没有影响，即使在用维拉帕米和/或丙磺舒抑制后也是如此。然而，HA 调控的 HBMEC 的双重培养促进了 P-gp 和 MRPs 的数量，并减少了 calcein-AM 的内吞作用。抑制和内吞分析可以揭示 HA 在 HBMEC 上的蛋白表达中的作用，以建立可靠的体外 BBB 模型。
