用Dextran T-70和蔗糖分离猪血脑屏障微血管的效果比较

Isolation and purification of micro-vessicles from porcine blood brain barrier with Dextran T-70 and sucrose

以DextranT 70和蔗糖为分离液 ,经组织匀浆、差数和密度梯度离心、玻璃柱色层过滤等步骤 ,从猪大脑组织中分离得到脑微血管内皮细胞 ,建立体外血脑屏障研究模型 ,并用显微分析技术和测定γ 谷氨酰转肽酶、 5′ 核苷酸酶、乙酰胆碱酯酶、乳酸脱氢酶等标志酶活性 ,鉴定该模型的纯化程度和完整性及生物学活性。结果表明 ,用两种分离液均能分离得到完整性好、纯度和生物学活性均高的微血管内皮细胞 ,尤其是用DextranT 70制备的微血管 ,其生物学活性和纯度高于用蔗糖离心所得的微血管。这些制备物可用于研究血脑屏障的代谢、结构。

In this experiment,two chemicals of Dextran T 70 and sucrose were used to isolate and purify brain endothelial cells of porcine cerebra via homogenization,differential and density gradient centrifugation,and chromatography on a column of glass beads,for construction of an in vitro blood brain barrier(BBB)model.The purity and biological activity of the preparation were monitored by microscopy and detection of the activity of such enzymes as γ glutamy transpeptidase(γ GTase),5′ nucleotidase(NTDase),acetylcholinesterase(ACSase),lactate dehydrogenase(LDH)and alkaline phosphatase(AKP).The results indicated that both chemicals could be used to successfully isolate biological activity of microvessicles with high purity.Especially the preparations purified with Dextran T 70 had a higher biological activity and purity than those purified with sucrose.This product provide a useful blood brain barrier model for the investigation of the regulating mechanism of different reproductive hormones and other growth factors which function through the central neural system.