猪脑微血管内细胞的原代分离培养与鉴定  

Primary Isolation,Culture and Idendtification of Pig Cerebral Microvascular Endothelial Cells

作　　者：赵春雨[1,2] 孔志伟[3] 张天芮 杨文艳[1,2] 杨连玉[1,2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]吉林省动物营养与饲料科学重点实验室,吉林长春130118 [3]中国科学院亚热带农业生态研究所,湖南长沙410125

出　　处：《中国兽医杂志》2015年第12期23-26,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金青年科学基金(31302002);吉林省教育厅项目(201252)

摘　　要：探讨获取纯度较高的原代猪脑微血管内皮细胞的分离和培养方法。采用1月龄的三元杂交猪,通过两次酶消化、BSA和Percoll非连续梯度离心获得较纯的脑微血管段后,接种于涂布有鼠尾胶的培养瓶进行原代培养;相差显微镜观察细胞并进行纯化和传代,采用Ⅷ因子相关抗原免疫荧光检测法对培养的细胞进行鉴定。结果表明,培养24 h即可见细胞从贴壁的脑微血管段周围爬出,细胞呈短梭形,集落呈典型的＂鹅卵石样＂,区域性单层生长,6~7 d内皮细胞开始融合,血管内皮细胞特异性标志物Ⅷ因子相关抗原表达阳性。说明本试验成功的培养出了纯度较高的脑微血管内皮细胞,为后续的体外血脑屏障模型的建立奠定了基础。This study was designed to develop an isolation and culture method to get higher purity of pig brain microvascular endothelial cells （BMEC）. After relatively pure cerebral mierovessel fragments were obtained from lmonth old Landraee by careful dissection, and two steps of enzyme digestions and gradient centrifugation with BSA and Pereoll were performed, the fragments were seeded on dishes coated with rattail collagen.PBMEC were identified according to the morphology of the cultured ceils and im munocytochemistry of factor VIII-associated antigen. We found that the cultured cells began to migrate from microvessel fragments after 24 hours cells showed the spindle-shaped morphology and looked like＂slabstene＂, ar typical appearance of cultured endothelial cells under inverted microscope and reached the monolayer confluence after6-7 days. Our cultured cells had factor VIII-associated antigen.The results indicated that relatively pure primary culture of PBMEC was successfully established.

关 键 词：猪 脑微血管内皮细胞 分离培养 免疫荧光 

分 类 号：S858.28[农业科学—临床兽医学]