永生化小鼠Bend.3细胞株具有脑微血管内皮细胞的屏障特性

Immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells

目的评价永生化小鼠脑微血管内皮细胞株Bend.3是否具有脑微血管内皮细胞的屏障特性。方法将小鼠脑微血管内皮细胞株Bend.3接种于细胞培养插内,跨内皮细胞电阻抗(transendothelial electrical resist-ance,TEER)和辣根过氧化物酶(horseradish peroxidase,HRP)通透性实验检测其屏障功能。Western blot法和直接荧光染色法观察其紧密连接相关蛋白Occludin、ZO-1的表达及细胞骨架蛋白F-actin的分布。结果 Bend.3细胞的TEER随培养时间延长逐渐升高,10d达82.3±6.0Ω.cm2,与培养3d时的37.3±3.1Ω.cm2相比,差异有统计学意义(P<0.05)。其培养10d和3d的平均HRP通透率在120min分别为(2.2±0.05)%和(4.3±0.20)%,差异有统计学意义(P<0.05)。培养10d时该细胞表达高浓度的紧密连接蛋白Occludin、ZO-1,且F-actin主要分布在细胞周边,线条完整连续,未见明显缝隙形成。结论小鼠脑微血管内皮细胞株Bend.3具有脑微血管内皮细胞的屏障特性,且其屏障功能在接种10d后可达到最理想的状态。

Objective The purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells（BEMC）.Methods Immortalized mouse brain endothelial cell line,Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance（TEER）and horseradish peroxidase（HRP）permeability assays.Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution.Results The TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3±6.0 Ω·cm2 10 days after culture,which was significantly higher than that 3 days after culture（37.3±3.1 Ω·cm2;P0.05）.There were significant differences in the permeability rates for HRP 3 and 10 days after culture [（4.3±0.20）% vs（2.2±0.05）%]（P0.05）.Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture.F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture.Conclusions Bend.3 cells have similar barrier characteristics to BEMC,and their barrier function may reach to the best effect 10 days after culture.