大鼠视网膜微血管内皮细胞的原代培养及鉴定改进

Improved primary culture and identification of rat retinal microvascular endothelial cells in vitro

目的探讨大鼠视网膜微血管内皮细胞原代培养改进方法,为研究视网膜新生血管疾病的病理机制奠定基础。设计实验研究。研究对象大鼠视网膜微血管内皮细胞。方法采用机械剪除大鼠视网膜血管联合酶消化分离,用含胎牛血清、内皮细胞培养特制添加剂等的条件培养基培养及纯化大鼠视网膜微血管内皮细胞,倒置相差显微镜下观察细胞生长状态。应用因子VIII、血小板内皮细胞黏附分子-1（CD31）免疫鉴定细胞。主要指标微血管内皮细胞形态,免疫荧光染色。结果原代培养的大鼠视网膜微血管内皮细胞贴壁后散在分布,呈单层排列,多为梭形,边界清楚,少量呈铺路石样螺旋向外生长,3-5天后融合,呈簇状单层分布。免疫荧光细胞染色显示,因子VIII抗体染色核周出现黄绿色荧光反应,CD31因子抗体染色以胞浆着色为主。细胞纯度较高,荧光染色阳性细胞率为95%。结论采用机械剪除大鼠视网膜血管、胰蛋白酶和胶原酶消化、含特制添加剂培养基的应用、明胶包被培养瓶等综合法可获得较纯的大鼠视网膜微血管内皮细胞。

Objective To establish a reliable method for primary culture of rat retinal microvascular endothelial cells （RRMEC） in vitro in order to study the basic pathogenesis of retinal neovascularization disease. Design Experimental study. Participants Rat retinal microvascular endothelial cells. Methods With the isolation of active retinal blood vessels and enzyme digestion of retinal tissue, RRMECs were cultured in RPMI-1640 supplemented with fetal bovine serum, and endothelial cell growth supplement （ECGs）. The RRMECs were observed under microscope. Immunocytochemistry was also performed using a monoclonal antibody against Factor VIII and platelet endothelial cell adhesion molecule （PECAM-1, CD31） for identification. Main Outcome Measures RRMEC morphology and fluorescence staining. Results RRMECs in primary culture attached the walls and scattered in monolayer arrangement with clear boundary, mostly in fusiform, partly in cobblestone-like growth. RRMECs began to confluence within 3-5 days. The purity of RRMEC was high. The rate of positive staining for Factor VIII （fluorescence staining perinuclear） and CD31 （fluorescence staining in cytoplasm） was 95%. Conclusions RRMECs with high purity can be obtained by isolation of retinal blood vessels, digestion with trypsin and colla- genase, and use of ECGs and gelatin-coated bottles, which is a repeatable better method. （Ophthalmol CHN, 2012, 21： 261-263）