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目的建立模拟体内血脑屏障(blood-brain barrier,BBB)模型,并运用该模型对丹酚酸组分中可能作用于中枢神经系统(CNS)的活性成分进行筛选,为CNS药物的快速高通量筛选提供实验依据。方法将人脑微血管内皮细胞(HBMEC)和人星形胶质细胞(HA)进行共培养,模拟体内BBB,建立细胞培养水平的模型,实时考察模型形成过程中细胞的跨膜电阻值(TEER)及通透性。并运用该模型对丹酚酸组分中可能作用于CNS的有效活性成分进行筛选,利用液质联用技术对筛选出的化合物进行初步鉴定。结果 HA诱导HBMEC产生较高的跨膜电阻,于第10天达300Ω/cm~2,荧光素钠检测内皮细胞形成的紧密连接,其跨体外BBB模型的渗透系数为(2.659±0.730)×10~(-3)cm/min。利用该模型从丹酚酸组分中筛选出至少有6个成分能够穿越BBB模型,这6个成分被确认为丹参素、原儿茶酸、咖啡酸、异阿魏酸、紫草酸、迷迭香酸。结论建立的体外模型在跨膜电阻和通透性方面具备了在体的基本特性,为CNS药物的快速、高通量筛选提供实验依据。
OBJECTIVE: To establish a model of blood-brain barrier (BBB) in vivo and use this model to screen active components of salvianolic acid that may act on the central nervous system (CNS) Screening provides experimental evidence. Methods Human BBM and human astrocyte (HA) were co-cultured to simulate BBB in vivo and to establish a model of cell culture. The transmembrane resistance (TEER) And permeability. The model was used to screen active constituents that may act on CNS in salvianolic acid. The compounds were identified by LC-MS. Results HA induced a high transmembrane resistance of HBMEC, reaching 300Ω / cm ~ 2 on the 10th day. Fluorescein sodium detected the tight junctions formed by endothelial cells. The transmembrane permeability coefficient of transmembrane BBB model was (2.659 ± 0.730) × 10 ~ (-3) cm / min. Using this model, at least six components were selected from the salvianolic acid component to cross the BBB model, and these six components were identified as Danshensu, Protocatechuic acid, Caffeic acid, Isoferulic acid, Lithospermin, Balsamic acid. Conclusion The established in vitro model possesses the basic characteristics of the transmembrane resistance and permeability in vivo and provides an experimental basis for rapid and high-throughput screening of CNS drugs.