血脑屏障体外实验模型的建立

Establishment of an in vitro model of brain-blood barrier

目的 :应用培养的CBA/J小鼠脑血管内皮细胞 (brainmicrovascularendothelialcells,BMVEC)构建血脑屏障(blood brainbarrier,BBB)的体外实验模型。方法 :将BMVEC种植在明胶包被的 2 4孔板细胞插入器的微孔滤膜上培养至汇合状态 ,通过 4h液面渗漏实验、扫描和透射电镜、血脑屏障形成前后膜两侧的电阻以及辣根过氧化物酶(horseradishperoxidase ,HRP)的通透性和RMP 7对血脑屏障通透性的调控作用来证实BBB的形成。结果 :BMVEC培养至汇合后 ,4h液面渗漏实验成为阳性 ;扫描电镜显示细胞形成单层 ,透射电镜证实细胞间形成紧密连接 ;跨细胞电阻 (thetransendothelialelectricalresistance ,TEER)分别为汇合前和人脐静脉内皮细胞的 3.2倍和 7.7倍 ;对HRP的通透率分别为前对照组的 1 3.4 %和 6 .7% ;RMP 7处理使HRP在BBB的通透率增加了 2 .7倍。结论 :构建的BBB体外模型在形态学、电阻和通透性方面具备了BBB的基本特性 。

Objective: To establish an in vitro model of brain blood barrier (BBB) using cultured mouse brain microvascular endothelial cells (BMVEC). Methods: Mouse BMVEC were seeded on micro pore membrane of gelatin coated cell culture insert and cultured to confluence. The establishment of BBB was preliminary judged by a 4 h water leaking test. The tight junctions between BMVEC were demonstrated by scanning and transmission electron microscope. The transendothelial electrical resistance(TEER) over BMVEC was measured. The permeability of Horseradish peroxidase (HRP) through the BBB was analyzed and the effect of RMP 7 on permeability of the BBB was investigated. Results: The 4 h water leaking test became positive when BMVEC were cultured to confluence. By scanning and transmission electron microscope, the tight junctions were demonstrated on confluent BMVEC. The TEER over BMVEC monolayer increased 3.2 and 7.68 times and the permeability rates for HRP were 13.4% and 6.7% respectively, as compared with sub confluent BMVEC and human umbilical vein endothelial cell monolayer(HUVEC). The HRP permeability rate in the model of BBB increased 2.7 times after treatment with RMP 7. Conclusion: The established in vitro model of BBB has basic characteristics of BBB in vivo , and is suitable for central nervous system (CNS) drug research over BBB.