大鼠脑微血管内皮细胞的分离与原代培养

为了建立大鼠脑微血管内皮细胞体外培养模型，探索纯度较高的大鼠脑微血管内皮细胞分离和原代培养的方法并进行形态学观察。采用2～3周龄的SD大鼠，解剖得到大脑皮质，两次酶消化及牛血清白蛋白或葡聚糖和Percoll梯度离心获得较纯的脑微血管段后，接种于涂布基质的培养皿进行原代培养；培养的细胞采用相差显微镜形态学观察、透射电镜观察及Ⅷ因子相关抗原免疫组化检测鉴定。结果发现，培养12h即可见细胞从贴壁的脑微血管段周围长出，细胞呈短梭形，区域性单层生长，5～7天内皮细胞融合，内皮细胞纯度达90％以上；内皮细胞的贴壁和生长有赖于所涂布的基质，纤连蛋白／Ⅳ型胶原优于鼠尾胶和明胶；Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性，透射电镜观察可见相邻内皮细胞间存在紧密连接结构。提示该方法能成功进行纯度较高的大鼠脑微血管内皮细胞原代培养，可用于脑微血管内皮的生理、生化及药理学研究，亦可用于构建大鼠血脑屏障模型。

Isolation and Primary Culture of Rat Cerebral Microvascular Endothelial Cells

To establish rat cerebral microvascular endothelial cells (RCMEC) culture model, we developeda method for isolation and primary culture of RCMEC and observed the morphology of endothelial cells. Afterrelatively pure cerebral microvessel fragments were obtained from 2-3 weeks old SD rats by careful dissection, twosteps of enzyme digestions and gradient centrifugation with BSA or dextran and Percoll, they were seeded on dishescoated with the substrata. RCMEC were identified according to the morphology of the cultured cells, immunocy-tochemistry of factor VIII-associated antigen and transmission electron microscopy. We found that the culturedcells began to migrate from microvessel fragments after 12 hours, showed the spindle-shaped morphology andreached the monolayer confluence after 5-7 days. Attachment and growth of the cultured cells depended on thesubstrata provided and fibronectin/type IV collagen was superior to collagen from rat tail and gelatin. The culturedcells had factor VIII-associated antigen and showed tight junction-like cell-cell appositions at the electron micro-scopic level. The results indicated that relatively pure primary culture of RCMEC was successfully established usingthis method and the model system could be applicable to the studies of physiology, biochemistry and pharmacologyof the brain endothelium and could also be used to develop an in vitro model of the rat blood-brain barrier.