大鼠脑皮质微血管内皮细胞的原代培养和鉴定

摘要

Objective To isolate,culture and identify rat brain microvascular endothelial cells(BMECs) ,so as to build up a method by which high purity and high yield of BMECs can be obtained. Methods We used homogenization, double enzyme digestion and gradient centrifugation to isolate microvessels of the brain in 3- 5-days old rats. Cells were cultured in culture plates coated with gelatin. The BMECs were observed under an inverted phase contrast microscope and identified by factor Ⅶ related antigen through immunohistochemistry. We determined the growth curve of BMECs by MTT assay. Results The BMECs were in spindle or polygon,and retained cobblestone appearance at about 6th-7th day. The factor Ⅶ related antigen was positive by immunofluorescent staining, and the purity was up to ( 96. 70 ± 1. 53 ) % . BMECs in the 6th - 8th day achieved growth peak. Conclusion Rat BMECs with high purity were successfully isolated and cultured in our study, which will supply great help for further research on the biological characteristics of BMECs.%目的 分离、培养、鉴定大脑皮质微血管内皮细胞(brain microvaseular endothelial cells,BMECs),旨在建立一种可以有效获取较高纯度和产量的BMECs的方法.方法 取出生3～5 dSD大鼠,采用匀浆、二次酶消化、梯度离心获得较纯的脑微血管段后,接种于明胶包被的培养板中进行原代培养,倒置相差显微镜观察细胞的形态学特性,细胞免疫荧光化学染色检测血管内皮细胞特异性标志物Ⅷ因子相关抗原的表达,噻唑蓝(MTT)比色法测定细胞生长曲线.结果 培养6～7 d细胞呈典型的短梭形、多角形和“鹅卵石”样.微血管内皮细胞特异性标志物Ⅷ因子相关抗原表达阳性,纯度达(96.70±1.53)％.细胞在6～8 d达到生长高峰.结论 成功地分离培养出了高纯度的BMECs,为进一步研究BMECs的生物学特性奠定了基础.