【摘要】 目的探讨获取纯度较高的原代大鼠脑微血管内皮细胞(BMECs)的分离和培养方法。方法选取10只3周龄Wistar大鼠,取大脑,去除白质,剪碎成大约1 mm~3大小,通过两次酶消化、20%BSA离心和33%连续密度Percoll梯度离心,获得纯度较高的大脑微血管片段,接种于涂布明胶的35 mm dish培养皿中,加入含4μg/ml嘌呤霉素的内皮细胞培养基,2d后更换新鲜培养基;采用倒置显微镜观察内皮细胞生长状况及其形态;免疫组化法检测BMECs标志物Ⅷ因子相关抗原(vWF)的表达,以及神经胶质原纤维酸性蛋白(GFAP)的表达情况。结果培养24 h后可见内皮细胞从贴壁的脑微血管片段周围长出,细胞呈梭形;5～6 d可形成融合;融合后的BMECs呈典型的"鹅卵石"样外观;免疫组化法检测显示BMECs的vWF表达阳性,CFAP表达阴性,内皮细胞纯度(vWF阳性细胞表达率)达96%以上。结论该方法能够成功获得纯度较高的原代大鼠BMECs。更多还原

【Abstract】 Objective To explore and obtain the isolation and culture methods of the highly purified primary rat brain microvascular endothelial cells (BMECs).Methods The brains of 10 3-week-old Wistar rats were harvested by decapitation,removing the white matter and mincing into approximately 1 mm~3.A highly purified brain microvascular fragments were obtained through enzymatic digestion twice, 20% bovine serum albumin (BSA) and 33% continuous Percoll density gradient eentrifugation.They were incubated into gelatin-coated 35 mm dish plates and the endothelial medium containing 4 μg/ml puromycins was added.The medium was renewed after 2 days.The endothelial cell growth and its morphology were observed using an inverted microscope.The expression of BMECs markers factor VⅢ-related antigen (vWF) was detected by immunocytochemical method and was identified.Results The endothelial cells grew out around the adherent brain microvascular fragments after culturing for 24 hours,and the cells were spindleshaped. A fusion was formed after 5 to 6 days.BMECs after the fusion showed a typical " cobblestone-like" appearance.Immunocytochemical analysis showed the vWF expression of BMECs was positive,while the glial fibrillary protein (GFAP) expression was negative.The purity of BMVECs reached more than 96%. Conclusion This method may successfully obtain the highly purified rat primary BMECs.更多还原