摘要
目的 建立一种程序简便、经济、获得细胞纯度较高的体外分离、培养大鼠脑微血管内皮细胞(BMECs)的方法。方法 以SD大鼠为实验材料,采用两步滤过法获得微血管段,胶原酶消化,低分子右旋糖苷密度离心获得BMECs,接种后4 h换液使获得的细胞纯化。倒置显微镜下观察细胞的形态,细胞Ⅷ因子相关抗原(ⅧF-Ag)免疫细胞化学法鉴定细胞及其纯度,通过碱性磷酸酶(AKP)的表达来分析BMECs被微动脉和微静脉污染的程度,通过测定BMECs分泌ET-1的量,鉴定培养的BMECs活力。结果 经分离培养获得的BMECs在倒置显微镜下呈多角形或"铺路石"形,单层贴壁生长。培养的细胞Ⅷ因子相关抗原免疫组化试验阳性,细胞纯度为90％。AKP染色阳性细胞百分比为93％,总积分112。原代培养内皮细胞的ET-1含量为388.03 pg／ml,传代后为591.75 pg／ml。结论 采用两步滤过法获得脑微血管段,胶原酶消化,低分子量右旋糖苷密度离心可分离和培养大鼠BMECs。该方法较其他方法程序简单,获得细胞纯度高。
Objective This study was to establish a method to isolate and culture brain microvas-cular endothelial cells(BMECs) in vitro. Methods BMECs were isolated from neonatal Sprague Daw-ley rats by two-step filtration, collagenase digestion and density gradient centrifugation using low molecular weight dextran. After 4 hour seeding, the floating cells were washed away and the adherant BMECs were kept. The cell morphology was observed under phase-contrast microscope. The purity of BMECs was examined by immunohistochemical staining. The contamination of microvessles was determined by the expression of AKP by BMECs. The viability of BMECs was determined by ET-1 releasing. Results The purity of cultured BMECs was 90% using this modified method. Polygonal or cobblestone like cells were observed under microscope, and cells are adherent, proliferated in a monolayer. The cultured BMECs were positive for ⅧF-Ag with immunohistochemical staining. The purity of positive stained cells was 90 % . The percentage of AKP-positive cells was 93 % , with a total score of 112. The primary BMECs released 388.03 pg/ml of ET-1 and the passage 2 cells secreted 591.75 pg/ml. Conclusion The rat BMECs were successfully cultrued in vitro by two-step filtration, collagenase digestion and density gradient centrifugation in our study. This method of isolation and culture of BMECs is easier and more efficient than others.
出处
《苏州大学学报（医学版）》
CAS
2004年第5期657-660,共4页
Suzhou University Journal of Medical Science
关键词
脑微血管内皮细胞
细胞培养
纯度
活力
brain microvascular endothelial cells
culture
purity
vitality
