King's College London, Institute of Pharmaceutical Science, BBB Group, Franklin Wilkins Building, 150 Stamford St, London SE1 9NH, UK.
Brain Res. 2013 Jul 12;1521:1-15. doi: 10.1016/j.brainres.2012.06.057. Epub 2012 Jul 10.
Good in vitro blood-brain barrier (BBB) models that mimic the in vivo BBB phenotype are essential for studies on BBB functionality and for initial screening in drug discovery programmes, as many potential therapeutic drug candidates have poor BBB permeation. Difficulties associated with the availability of human brain tissue, coupled with the time and cost associated with using animals for this kind of research have led to the development of non-human cell culture models. However, most BBB models display a low transendothelial electrical resistance (TEER), which is a measure of the tightness of the BBB. To address these issues we have established and optimised a robust, simple to use in vitro BBB model using porcine brain endothelial cells (PBECs). The PBEC model gives high TEER without the need for co-culture with astrocytes (up to 1300 O cm(2) with a mean TEER of ~800 O cm(2)) with well organised tight junctions as shown by immunostaining for occludin and claudin-5. Functional assays confirmed the presence of high levels of alkaline phosphatase (ALP), and presence of the efflux transporter, P-glycoprotein (P-gp, ABCB1). Presence of the breast cancer resistance protein (BCRP, ABCG2) was confirmed by TaqMan real-time RT-PCR assay. Real-time RT-PCR assays for BCRP, occludin and claudin-5 demonstrated no significant differences between batches of PBECs, and also between primary and passage 1 PBECs. A permeability screen of 10 compounds demonstrated the usefulness of the model as a tool for drug permeability studies. Qualitative and quantitative results from this study confirm that this in vitro porcine BBB model is reliable and robust; it is also simpler to generate than most other BBB models. This article is part of a Special Issue entitled Electrical Synapses.
好的，体外血脑屏障 (BBB) 模型可以模拟体内 BBB 表型，对于研究 BBB 功能和药物发现计划的初步筛选非常重要，因为许多潜在的治疗候选药物的 BBB 通透性较差。由于人脑组织的可用性存在困难，再加上使用动物进行此类研究的时间和成本，因此开发了非人类细胞培养模型。然而，大多数 BBB 模型的跨内皮电阻 (TEER) 较低，TEER 是衡量 BBB 紧密程度的指标。为了解决这些问题，我们使用猪脑内皮细胞 (PBEC) 建立并优化了一种稳健、易于使用的体外 BBB 模型。该 PBEC 模型在无需与星形胶质细胞共培养的情况下即可获得高 TEER（高达 1300 O cm(2)，平均 TEER 约为 800 O cm(2)），紧密连接排列良好，如用 occludin 和 claudin-5 免疫染色所示。功能测定证实碱性磷酸酶 (ALP) 水平较高，并且存在外排转运蛋白 P-糖蛋白 (P-gp，ABCB1)。通过 TaqMan 实时 RT-PCR 测定法证实存在乳腺癌耐药蛋白 (BCRP，ABCG2)。BCRP、occludin 和 claudin-5 的实时 RT-PCR 测定表明 PBEC 批次之间以及原代和第 1 代 PBEC 之间没有显着差异。对 10 种化合物的通透性筛选表明该模型是药物通透性研究的有用工具。来自该研究的定性和定量结果证实了这种体外猪 BBB 模型的可靠性和稳健性；它也比大多数其他 BBB 模型更容易生成。本文是特刊题为“电突触”的一部分。
