改良原代大鼠脑微血管内皮细胞的培养方法及鉴定

目的 改良原代大鼠脑微血管内皮细胞分离培养方法。方法 选取4～6周龄SD大鼠6只，经开颅取脑、漂洗剪碎、过筛、牛血清白蛋白密度梯度离心、Ⅱ型胶原酶及胶原酶-分散酶两次连续酶消化后进行原代培养。通过细胞形态学观察和第Ⅷ因子免疫细胞化学染色鉴定所培养的目的细胞。结果 体外培养12～24 h后，细胞以贴壁的脑微血管段为中心，放射状向外周移行，并逐渐扩大成团簇状；细胞融合后则呈典型的单层、扁平、“铺路石样”镶嵌式排列。第Ⅷ因子免疫细胞化学染色检测，胞质呈棕红色，表达为阳性，阳性细胞率达99％以上。结论 改良方法能够成功高效分离培养出原代大鼠脑微血管内皮细胞。

Improvement on the methods about the primary culture of rat brain microvascular endothelial cells and identification

Objective To improve the method about the primary culture of rat brain microvascular endothelial cells in vitro.Methods The SD rats aged from 4 to 6 weeks were chosen as research object. After craniotomy, washing and cutting, sieving, density gradient concentration of BSA, digestion of type Ⅱ collagenase and collagenase dispersive enzyme twice, the primary culture was carried out. The target cells were indentified by morphological abservation and immunocytochemical staining of facter Ⅷ.Results Cultured for 12 to 24 hours，the cells in vitro migrated outward from the microvascular section. The cells appeared polygonal-shaped，and proliferated in a clustered monolayer. the cell growth density reached 70％-80％ of the bottle bottom after 3 days，and arranged like cobbles. The correlation antigen of Ⅷ factor was positive，they reached confluence with over purity 99％.Conclusion The method is available that can successfully separate and cultivate microvascular endothelial cells of rat brains in vitro.