两次过滤体外分离培养原代血脑屏障内皮细胞  

In vitro isolation and culture of primary blood-brain barrier endothelial cells by double filtering technique

作　　者：刘洁晓[1] 蒲传强[1] 郝延磊[1] 

机构地区：[1]解放军总医院神经内科,北京市100853

出　　处：《中国组织工程研究与临床康复》2008年第21期4123-4126,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(39670267)~~

摘　　要：背景：内皮细胞是血脑屏障结构和功能的主要基础，要了解血脑屏障的作用，就必须在体外建立血脑屏障模型。目的：利用幼鼠脑微血管片段模拟血脑屏障内皮细胞的生物学特性，拟建立一种血脑屏障内皮细胞的体外培养技术。设计、时间及地点：细胞观察，于2006—04在解放军总医院神经内科实验室完成。材料：清洁级1月龄SD大鼠2只，用于脑组织微血管内皮细胞的制备。方法：取大鼠脑组织，加入D—Hank’s液培养基制备匀浆，149μm滤网过滤后收集滤液，以74μm滤网再次过滤。将滤网上的组织刮下，胶原酶消化后传代扩增。取传1代后的脑组织微血管内皮细胞用于指标检测。主要观察指标：细胞形态变化。锥虫蓝染色计数细胞活性。免疫组织化学染色鉴定。结果：①培养12h后大部分脑微血管片段贴壁，两三天微血管片段周边有成簇的细胞团及单个细胞生长，7～10d细胞生长为致密单层并呈典型鹅卵石样排列。传代细胞4h开始贴壁，培养一两天变为多角形并呈致密单层。②生长7～10d的细胞其活性〉96％。③培养的细胞第Ⅷ因子多克隆抗体免疫组化染色显示胞膜和胞浆呈阳性，胞核为阴性，为血脑屏障内皮细胞。结论：采用两次过滤培养技术可成功分离培养出较理想的原代血脑屏障内皮细胞。BACKGROUND： Endothelial cells are the basis of structure and function of blood-brain barrier. To understand the effect of blood-brain barrier, we should establish a blood-brain barrier model in vitro. OBJECTIVE： To investigate techniques of in vitro culture and identification of endothelial cells of blood-brain barrier by simulating biological feature of blood-brain barrier endothelial cells with brain capillary vessel segments in young rats. DESIGN, TIME AND SETTING： A cell observational study was performed at the Laboratory of Department of Neurology, General Hospital of Chinese PLA in April, 2006. MATERIALS： Two clean SD rats aged 1 month were used to prepare vascular endothelial cells of brain tissues. METHODS： Rat brain tissues were collected to prepare homogenate in D-Hank＇s medium. Filter liquor was collected after filtering with a 149 μ m filter, and then filtered with a 74μ m filter. Tissues on the filter were collected and amplified with collagenase digestion. After first passage, microvascular endothelial cells of brain tissues were measured. MAIN OUTCOME MEASURES： Changes in cell morphology; Cell activity was detected by trypan blue staining and determined by immunohistochemistry. RESULTS： In 12 hours after cultivation, segment of cerebral microvessels adhered to the wall. In 2-3 days, cluster or single cells were around segment of cerebral microvessels. In 7 10 days, the cultural cells arranged as ovum-like in compact monolayer. Subcultured cells grew in 4 hours and became polygon arranging as single layer in 1 2 days. Activity of cells was 〉 96% 7- 10 days after culture. Immunohistochemistry of Ⅷ factor polyclone antibody showed positive in cytomembrane and cytoplasm, but negative in cell nucleus. These were cells with blood-brain barrier. CONCLUSION： Ideal endothelial cells of blood-brain barrier can be cultured by the improved cultural method of double filtering.

关 键 词：内皮细胞 细胞培养 血脑屏障 

分 类 号：R394.2[医药卫生—医学遗传学]