Blood-brain barrier functioning as dynamic interface and drug delivery to the brain

本研究的目的是通过调节载体介导的营养物质和外源物的流入和流出来阐明血脑屏障（BBB）的功能。本课题历时两年，取得了以下研究成果：在短暂性脑缺血大鼠中，脑内ATP耗竭导致阿霉素（DOX）血脑屏障通透系数（PS）增加17倍，而蔗糖的PS值未因缺血而改变。当ATP通过脑血液再循环恢复到正常水平时，DOX的ps值恢复到正常大鼠的对照ps值。原代培养的牛脑毛细血管内皮细胞（BCECs）在管腔膜上表达p -糖蛋白（P-gp），通过消耗BCECs内ATP水平显著增加DOX的摄取。目前关于DOX的atp依赖性运输的证据和我们实验室之前的结果强烈表明，脑毛细血管中的P-gp作为亲脂性外排泵积极发挥作用，提供了限制外源药物和DOX在脑中的转移的主要机制2。β -丙氨酸和牛磺酸的二级主动转运系统以钠离子和氯离子依赖的方式运输，在血脑屏障的管腔膜和抗管腔膜上都起作用。转运系统对β -氨基酸，如-丙氨酸、牛磺酸和次牛磺酸具有高度选择性。RT-PCR分析了H^+/单羧酸转运体MCT1在脑毛细血管中的表达，并证实PCR产物的核苷酸序列与部分大鼠MCT1的核苷酸序列一致。这一结果表明MCT1参与了ph依赖性和载体介导的血脑屏障单羧酸的内流和外排。多肽通过吸附介导的内吞作用被bcec吸收，这一作用受多肽的电荷密度和/或亲脂性的影响。

The purpose of this research project is to clarify the blood-brain barrier (BBB) functions by regulating carrier-mediated influx and efflux of nutrients and xenobiotics. The following results were obtained by two years term of this research project :1.In transient brain ischemic rats, ATP depletion in the brain resulted in the 17-fold increase of BBB permeability coefficient (PS) of doxorubicin (DOX), whereas no change of PS-value of sucrose by this ischemia. When the ATP content recovered to a normal level by means of cerebral recirculation of blood, the PS-value of DOX recovered to the control PS-value in normal rats. The uptake of DOX by primary cultured bovine brain capillary endothelial cells (BCECs) expressing P-glycoprotein (P-gp) at the luminal membrane was increased significantly by the depletion of ATP level in BCECs. Present evidence for the ATP-dependent transport of DOX and previous results from our laboratory strongly indicate that P-gp in the brain capillaries functions actively as an efflux pump for lipophilic xenobiotics, providing a major mechanism to restrict the transfer of xenobiotics as well as DOX in the brain.2.Secondary active transport system for beta-alanine and taurine, which are transported in sodium and chloride-ion dependent manner, functions at both the luminal and antiluminal membranes of the BBB.The transport system was highly selective for beta-amino acids, such as beta-alanine, taurine and hypotaurine.3.The expression of H^+/monocarboxylate transporter MCT1 in the brain capillaries was clarified by RT-PCR and the nucleotide sequence of the PCR product was confirmed to be the same as that of a part of ratMCT1. This result indicates that MCT1 contributes to pH-dependent and carrier-mediated influx and efflux of monocarboxylic acids at the BBB.4.Peptides were confirmed to be taken up by BCECs via adsorptive-mediated endocytosis which was affected by charge density and/or lipophilicity of peptides.