血脑屏障模型的建立及药物转运分析-establishment of blood-brain barrier model and analysis of drug transport.docxVIP

杏内酯 A 裸药的通透系数没有发生明显的变化。结论：1．通过 ECV304 细胞与星形胶质细胞共培养在涂有左旋多聚赖氨酸 的微孔膜两侧，能够形成与在体血脑屏障类似的结构，具有良好的酶屏障和限制 物质通透功能。这一共培养模型可以作为研究血脑屏障结构与功能的一种可靠而 简便的体外实验方法，适合用于血脑屏障物质转运和细胞功能特征方面的研究； 2．银杏内酯 A 的聚乳酸纳米粒和固体脂质体可以在短暂时间内增加体外血脑屏 障模型的通透性，说明有利于目的药物穿透血脑屏障。关键词：血脑屏障；胶质细胞；共培养；紧密连接；银杏内酯 A；药物转运AbstractA well-controlled internal environment is crucial for the function of the central nervous system. The mechanism that maintains the homeostasis of the central nervous system within the brain is known as blood-brain barrier, it is restricted for drugs to enter the tissue of brain because of the existence of BBB. In this research, an in vitro blood-brain barrier model was established by co-culturing endothelial cell and glial cell, moreover the structure and function were investigated. Ginkgolide A(GA) was selected, its polylactic acid nanoparticle(PLA-NP) and solid lipid nanoparticle(SLN) was prepared respectively, whose permeability was evaluated using the model of in vitro BBB.Methods: 1.An in vitro blood-brain barrier model was established by co-culturing endothelial cell(ECV-304) and glial cell(astrocytes or glioma cells); 2.Transendothelial electrical resistance of in vitro BBB model was studied by Millipore-ERS system; 3.Morphological change of tight junctions in the blood-brain barrier was detected by silver staining; 4.The activity of γ-L-glutamyl transpeptidase(γ-GT) and alkaline phosphatase(ALP) were measured with diagnostic assay kits; 5.Restriction of paracellular transport was determined by measurement of the apparent permeability co-efficient(Pe) for fluorescein sodium(Flu); 6.The permeability of GA, GA-PLA-NP and GA-SLN were evaluated using the model of in vitro BBB, HPLC-ELSD was used to determine the concentration of GA.Results: 1.After shaking culture, the purity of positive stained cells was 99%; 2. The in vitro BBB had a high TER value of 321.8±21.1Ω·cm2 after co-cultured with astrocytes for 6d, and the TER of BBB was decreased to 255.5±6.6Ω·cm2 after co-cultured with glioma cell