Background The complexity of the neurovascular unit (NVU) poses a challenge in the investigations of drug transport across the blood–brain barrier (BBB) and the function of the brain capillary endothelium. Several in vitro models of the brain capillary endothelium have been developed. In vitro culture of primary endothelial cells has, however, been reported to alter the expression levels of various brain endothelial proteins. Only a limited number of studies have addressed this in detail. The aim of the present study was to investigate mRNA levels of selected BBB transporters and markers in in vitro models of the BBB based on bovine primary endothelial cells and compare these to the levels estimated in freshly isolated bovine brain capillaries. Methods Brain capillaries were isolated from bovine cerebral cortex grey matter. Capillaries were seeded in culture flasks and endothelial cells were obtained using a brief trypsinization. They were seeded onto permeable supports and cultured in mono-, non-contact- or contact co-culture with/without primary rat astrocytes. mRNA-expression levels of the selected BBB markers and transporters were evaluated using qPCR and monolayer integrity of resulting monolayers was evaluated by measuring the transendothelial electrical resistance (TEER). Results The capillary mRNA transcript profile indicated low expression of ABCC1 and CLDN1 . The mRNA expression levels of TPA , OCLN , ABCB1 , SLC2A1 , SLC16A1 and SLC7A5 were significantly decreased in all culture configurations compared to freshly isolated bovine brain capillaries. ALP , VWF , ABCC1 and ABCC4 were upregulated during culture, while the mRNA expression levels of F11R , TJP1 , CLDN5 , CLDN1 and ABCG2 were found to be unaltered. The mRNA expression levels of VWF , ALP , ABCB1 and ABCC1 were affected by the presence of rat astrocytes. Conclusion The endothelial mRNA transcript profile in bovine capillaries obtained in this study correlated nicely with profiles reported in mice and humans. Cultured endothelial cells drastically downregulated the mRNA expression of the investigated SLC transporters but maintained expression of efflux transporter and junctional protein mRNA, implying that the bovine in vitro BBB models may serve well to investigate basic barrier biology and in vivo permeation of passively permeating compounds and efflux transporter substrates but may be less well suited for investigations of SLC-mediated transport.

中文翻译：

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培养诱导的脑内皮细胞外排和 SLC 转运蛋白 mRNA 表达水平的变化

背景 神经血管单元 (NVU) 的复杂性对跨血脑屏障 (BBB) 的药物转运和脑毛细血管内皮功能的研究提出了挑战。已经开发了几种脑毛细血管内皮的体外模型。然而，据报道，原代内皮细胞的体外培养会改变各种脑内皮蛋白的表达水平。只有有限数量的研究详细解决了这个问题。本研究的目的是在基于牛原代内皮细胞的 BBB 体外模型中研究所选 BBB 转运蛋白和标记物的 mRNA 水平，并将这些与新鲜分离的牛脑毛细血管中估计的水平进行比较。方法从牛大脑皮层灰质中分离出脑毛细血管。将毛细血管接种在培养瓶中，并使用简短的胰蛋白酶消化获得内皮细胞。它们被接种到可渗透的支持物上，并在有/没有原代大鼠星形胶质细胞的单、非接触或接触共培养中培养。使用 qPCR 评估所选 BBB 标记物和转运蛋白的 mRNA 表达水平，并通过测量跨内皮电阻 (TEER) 评估所得单层的单层完整性。结果毛细血管mRNA转录谱表明ABCC1和CLDN1的低表达。与新鲜分离的牛脑毛细血管相比，TPA、OCLN、ABCB1、SLC2A1、SLC16A1 和 SLC7A5 的 mRNA 表达水平在所有培养配置中均显着降低。ALP、VWF、ABCC1 和 ABCC4 在培养过程中上调，而 F11R、TJP1、发现CLDN5、CLDN1和ABCG2未改变。VWF、ALP、ABCB1和ABCC1的mRNA表达水平受大鼠星形胶质细胞存在的影响。结论 本研究中获得的牛毛细血管内皮 mRNA 转录谱与小鼠和人类报道的谱很好地相关。培养的内皮细胞显着下调所研究的 SLC 转运蛋白的 mRNA 表达，但保持外排转运蛋白和连接蛋白 mRNA 的表达，这意味着牛体外 BBB 模型可以很好地研究基本屏障生物学和被动渗透化合物的体内渗透和流出转运底物，但可能不太适合 SLC 介导的转运研究。ABCB1 和 ABCC1 受大鼠星形胶质细胞的影响。结论 本研究中获得的牛毛细血管内皮 mRNA 转录谱与小鼠和人类报道的谱很好地相关。培养的内皮细胞显着下调所研究的 SLC 转运蛋白的 mRNA 表达，但维持外排转运蛋白和连接蛋白 mRNA 的表达，这意味着牛体外 BBB 模型可以很好地研究基本屏障生物学和被动渗透化合物的体内渗透和流出转运底物，但可能不太适合 SLC 介导的转运研究。ABCB1 和 ABCC1 受大鼠星形胶质细胞的影响。结论 本研究中获得的牛毛细血管内皮 mRNA 转录谱与小鼠和人类报道的谱很好地相关。培养的内皮细胞显着下调所研究的 SLC 转运蛋白的 mRNA 表达，但维持外排转运蛋白和连接蛋白 mRNA 的表达，这意味着牛体外 BBB 模型可以很好地研究基本屏障生物学和被动渗透化合物的体内渗透和流出转运底物，但可能不太适合 SLC 介导的转运研究。结论 本研究中获得的牛毛细血管内皮 mRNA 转录谱与小鼠和人类报道的谱很好地相关。培养的内皮细胞显着下调所研究的 SLC 转运蛋白的 mRNA 表达，但维持外排转运蛋白和连接蛋白 mRNA 的表达，这意味着牛体外 BBB 模型可以很好地研究基本屏障生物学和被动渗透化合物的体内渗透和流出转运底物，但可能不太适合 SLC 介导的转运研究。结论 本研究中获得的牛毛细血管内皮 mRNA 转录谱与小鼠和人类报道的谱很好地相关。培养的内皮细胞显着下调所研究的 SLC 转运蛋白的 mRNA 表达，但维持外排转运蛋白和连接蛋白 mRNA 的表达，这意味着牛体外 BBB 模型可以很好地研究基本屏障生物学和被动渗透化合物的体内渗透和流出转运底物，但可能不太适合 SLC 介导的转运研究。