体外血脑屏障模型的建立

Establishment of an in vitro model of the blood brain barrier

摘要:

目的：建立一种相对简单可靠、接近在体状态的SD大鼠体外血脑屏障模型。方法：原代分离、纯化培养大鼠脑微血管内皮细胞和星型胶质细胞,将两种细胞共培养建立体外血脑屏障模型。VIII因子、GFAP免疫组化鉴定细胞类型,相差显微镜下观察细胞形态,透射电镜观察内皮细胞超微结构及紧密连接,γ计数仪测定核素标记牛血清白蛋白(125I-BSA)通透量检测血脑屏障(BBB)功能。结果：VIII因子、GFAP免疫组织化学结果显示培养的脑微血管内皮细胞和星形胶质细胞纯度分别为90%和99%以上。透射电镜显示内皮细胞间有高度发达的紧密连接、未见有W eible-Palade小体、少见吞饮小泡1。25I-BSA通透量结果显示,血脑屏障模型组在限制大分子量物质的能力上优于其他各组,与脑微血管内皮细胞单独培养组及空白对照组比较,差异有显著性意义(P<0.01)。结论：利用同种属脑微血管内皮细胞与星型胶质细胞共培养,建立了一种简便可行、与在体血脑屏障结构类似,并具有在体血脑屏障的限制物质通透能力的SD大鼠体外血脑屏障模型。[中国当代儿科杂志,2005,7(6):526-529]

Abstract:

OBJECTIVE: To establish a relatively simple,reliable and reproducible in vitro model of the blood brain barrier (BBB). METHODS: Cortical brain microvascular endothelial cells (BMEC) and astrocytes (AS) from 7-day-old rats were co-cultured in cell culture inserts.VIII-factor and GFAP immunocytochemistry were used to identify the cell types of the BBB model. Cell morphological changes were observed under light and transmission electron microscopes.At the same time, the BBB restrictive characteristics were assessed by ~(125) I-BSA permeability. RESULTS: The positive rates of BMEC and AS were 95 % and 99%respectively. The densely packed cells were observed and the characteristic BMEC 'cobblestones' were exhibited under the microscope on the 10th day of culture. Meanwhile a relative lack of pinocytic vesicles and the tight junction between the BMEC were presented. BMEC displayed a significant restriction of paracellular transport. CONCLUSIONS: An in vitro BBB model by co-culture with allogenic BMEC with AS was established, which was simple, reliable and reproducible as well as similar to the in vivo BBB in respect to morphology, ultrastructures and restriction characteristics.