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

本研究的目的是阐明血脑屏障（BBB）的功能，通过调节载体介导的流入和流出的营养物质和外源性物质。本研究通过2年的实验，获得了以下结果：1.短暂性脑缺血大鼠，脑内ATP耗竭导致血脑屏障对阿霉素（DOX）的通透性增加17倍，而对蔗糖的通透性无明显影响。当ATP含量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）表达P-糖蛋白（P-gp），ATP耗竭可显著增加BCECs对阿霉素的摄取。目前的证据表明，DOX的ATP依赖性转运和我们实验室以前的结果强烈表明，脑毛细血管中的P-gp作为亲脂性外源性物质的外排泵发挥积极作用，提供了限制外源性物质以及DOX在脑中转运的主要机制。2. β-丙氨酸和牛磺酸的次级主动转运系统，它们以钠和氯离子依赖的方式转运，该转运系统对β-丙氨酸、牛磺酸和次牛磺酸等β-氨基酸具有高度的选择性。3.通过RT-PCR方法，明确了H^+/单羧酸转运蛋白MCT 1在脑毛细血管中的表达，并证实了PCR产物的核苷酸序列与大鼠MCT 1的部分核苷酸序列相同。这一结果表明，MCT 1参与了pH依赖性和载体介导的一元羧酸在BBB的内流和外流。4.证实了肽通过吸附介导的内吞作用被BCECs摄取，该作用受肽的电荷密度和/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.