Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies
Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies
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DOI：
10.1016/s0006-8993(03)03443-7
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发表时间：
2003-11-14
期刊：
影响因子：
2.9
通讯作者：
Gumbleton, M
中科院分区：
文献类型：
--
作者：
Omidi, Y;Campbell, L;Gumbleton, M
II-表征的细胞系代表了研究内源性溶质或异生物质转运的重要工具。从用多瘤病毒中间T抗原转化的小鼠中分离的脑微血管细胞系b.End3可商购获得。在这里，我们报告的特性的一些功能的b.End3的相关使用血脑屏障转运的调查。在培养中，b.End3细胞显示出独特的梭形鳞状形态。网格蛋白包被的坑和许多未涂覆的细胞内囊泡是明显的细胞内，是表达的囊泡相关蛋白，网格蛋白，小窝蛋白-1，flotillin和发动蛋白II。在C6星形胶质细胞共培养b.End3单层中，最大跨内皮电阻为130 Ohm cm（2），但对跨细胞和细胞旁探针的渗透缺乏真实的区分，例如普萘洛尔的渗透系数（x 10（-6）cm s（-1））约为23，蔗糖为16。RT-PCR分析证实在b.End3细胞内存在以下转运蛋白的mRNA转录物：GLUT-1; MCT 1和2; OAT 1; Oatp 1; mdr 1a和1b; MR-P I和5; 3-丙氨酸，系统L和系统y（+）L氨基酸载体;核苷转运蛋白cNT 1和2、eNT 1和2以及紧密连接元件ZO-1、JAM、闭合蛋白、claudin-1和-5。b.End3细胞以钠非依赖性方式主动积累D-葡萄糖，其特征与GLUT-1一致。通过罗丹明-123蓄积和保留试验评估，P-糖蛋白外排的功能是明显的。通过摄取L-亮氨酸和L-苯丙氨酸并提供K.，和Vi，a值分别约为16 μ M和350-480 pmol/mg蛋白质/10 min;当b.End3细胞在C6星形胶质细胞因子存在下生长时，对这些底物的转运亲和力较弱，约为3倍。虽然b.End3细胞似乎不适合跨内皮渗透性评估，但它们显示出的特性使其有价值地用于研究血脑屏障转运机制。(C)2003 Elsevier B. V.保留所有权利。
ll-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood-brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Ohm cm(2), but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients ( x 10(-6) cm s(-1)) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MR-P I and 5; 3-alanine, system L and system y(+)L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated D-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/ 4F2hc amino acid transporter was examined through uptake Of L-leucine and L-phenylalanine and provided K., and V,,a, values of approximately 16 muM and 350-480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood-brain barrier transport mechanisms. (C) 2003 Elsevier B.V. All rights reserved.