Control of the drug efflux transport from the brain utilizing the transport function for the large molecules〜

本研究的最终目的是控制药物外排转运蛋白在血脑屏障（BBB）中的表达，提高药物的脑转移性。通过在脑毛细血管内皮细胞中引入抗体和/或反义基因，有可能抑制外排转运体。然而，这些大分子不容易形成血脑屏障。一些生物活性肽和蛋白质通过血脑屏障的特殊运输系统被大脑异常地吸收。利用这一转运系统，反义基因可以被转运到脑毛细血管内皮细胞中。然而，关于血脑屏障中肽或蛋白质转运系统的信息有限。在本研究中，研究者首次阐明了血脑屏障中的硫酸肝素蛋白多糖（HSPG）对包括生物活性肽和蛋白质在内的大分子转运系统的功能。利用条件永生化小鼠脑毛细血管内皮细胞系（TM-EBB），进一步证实了HSPG核心蛋白小鼠perlecan基因在TM-BBB上的表达。接下来，使用^<125>l-bFGF作为HSPG的配体来检测HSPG的输运函数。^<125>l-bFGF与牛脑分离的脑毛细血管紧密结合。^<125>l-bFGF内化TM-BBB表现出浓度依赖性、温度依赖性和渗透压依赖性。此外，肝素、硫酸软骨素B的内化被显著抑制，肝素酶和氯酸钠处理的细胞和小鼠perlecan抗体的内化也被抑制。脑原位灌注^<125>的l-bFGF显示未改变的^<125>的l-bFGF摄取到脑实质。肝素共灌注显著抑制了^<125>l-bFGF的脑摄取。从这些结果可以清楚地看出，HSPG在血脑屏障中表达，并作为大分子到大脑的运输系统发挥作用。未来利用这种转运系统，控制血脑屏障外排转运蛋白功能的抗体和/或反义基因可能被有效地转运到脑内皮细胞中。少

The final goal of this study is to control the expression of drug efflux transporter in the blood-brain barrier (BBB) and to improve the brain transferability of the drug. There is the possibility of suppressing the efflux transporter by introducing the antibody and/or antisense gene in the brain capillary endothelial cell. However, these giant molecule does not BBB easily. Some bioactive peptides and proteins are exceptionally taken up by the brain through the specialized transportation system of the BBB.If such transport system is utilized, antisense gene may be delivered into the brain capillary endothelial cells. However, information on the peptide/or protein transport system in the BBB is limited, In this study, the researcher clarified for the first time that heparan sulfate proteoglycan (HSPG) of the BBB functions transport system of large molecules including bioactive peptides and proteins.Using conditionally immortalized mouse brain capillary endothelial cell lines (TM-EBB), i … More t was confirmed that the gene expression of mouse perlecan which is the core protein of HSPG is expressed at TM-BBB.Next, the transport function of HSPG is examined using ^<125>l-bFGF as a ligand to HSPG.^<125>l-bFGF avidly bound to the brain capillary isolated from the bovine brain. Internalization of ^<125>l-bFGF into TM-BBB showed concentration-dependence, temperature and osmolarity dependence. In addition, the internalization was significantly inhibited in heparin, chondroitin sulfate B, and it was also inhibited in cells treated with heparinase and sodium chlorate, and antibody to mouse perlecan. In situ brain perfusion of ^<125>l-bFGF revealed the uptake into the brain parenchyma of unchanged ^<125>l-bFGF.This brain uptake of ^<125>l-bFGF was significantly inhibited by the heparin co-perfusion. From these results, it became clear that HSPG is expressed in the BBB and that it functions as the transport system of large molecule to the brain. By utilizing this transport system in future, antibody and/or antisense gene, which controls the function of the efflux transporter in the BBB, may be efficiently delivered into the brain endothelial cells. Less