体外血脑屏障模型的建立与鉴定-第三军医大学学报.doc

体外血脑屏障模型的建立与鉴定 杨钰楠1,高云华1,娄晋宁2,刘 平1,付赤学1 (1第三军医大学新桥医院超声科, 重庆 400037; 2中日友好医院病理生理教研室, 北京 100083) 提 要:目的 应用CBA/J小鼠脑微血管内皮细胞(brain microvascular endothelial cells, BMVEC)建立体外血脑屏障模型,探讨最佳构建条件。方法 用CBA/J小鼠脑血管内皮细胞建立血脑屏障（BBB）体外实验模型，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电阻与辣根过氧化物酶(horseradish peroxidase , HRP) 的通透性的关系,以及紧密连接ZO-1蛋白免疫组化等方法，评价血脑屏障模型的建立及特性。 结果 BMVEC培养至汇合后, 4h 液面渗漏实验为阳性；扫描电镜显示细胞形成单层,透射电镜、ZO-1蛋白免疫组化证实细胞间形成紧密连接；HRP的通透率与实时电阻呈负相关，内皮细胞电阻随着通透性的增加而减低，通透率最低时跨细胞电阻为（346±10）Ω·cm2。 结论 建立的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。 关键词:血脑屏障；体外模型；通透率；电阻 Establishment of an in vitro model of brain blood barrier YANG Yu-nan1, GAO Yu-hua1, LOU Jin-ning2, LIU Ping1, FU Ci-xue1(1 Department of Ultrasound, Third Military Medical University, Chongqing, 400037; 2 Institute of Clinical Medical Sciences, China-Japan Freindship Hospital) Abstract: Objective To establish an in vitro model of brain blood barrier (BBB) using cultured mouse brain microvascular endothelial cells (BMVEC), to explore the best culture condition. 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 4h 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 immunohistochemistry of the ZO-1 protein was investigated. Results The 4h water leaking test became positive when BMVEC were cultured to confluence. By scanning and transmission electron microscope, also immunohistochemistry of the ZO-1 protein, the tight junctions were demonstrated on confluent BMVEC. The TEER over BMVEC monolayer increased to （346±10）Ω·cm2 when the permeability rates for HRP were decreased. Conclusion The established in vitro model of BBB has basic characteristics of BBB in vivo, and is suitable for the research of diff