牛视网膜微血管内皮细胞的体外分离培养

Primary Culture of Bovine Retinal Capillary Endothelial Cells and Pericytes in vitro

目的探讨一种方便可靠的牛视网膜微血管内皮细胞(borein retinal microvas cular endothelial cell,BREC)体外培养方法。方法采用机械剪碎牛视网膜血管,用含牛血清、肝素、血管内皮生长因子(Vascularendothelial growth factor,VEGF)的条件化培养基培养牛视网膜血管内皮细胞,观察细胞生长状况,制作生长曲线。应用 Von Willebrand 因子抗体免疫鉴定 BREC,并通过透射电镜观察 BREC 的超微结构。结果添加了 VEGF 的条件化培养基对 BREC 有很好的选择刺激作用,初期细胞呈鲤鱼样聚集在微血管碎片周围,对数生长期细胞似铺路石样大片单层生长,存在接触抑制。Von Willebrand 因子抗体染色阳性,获得 BREC 纯度在95%以上,能够连续传代。培养的细胞电镜下符合内皮细胞的特征。结论含 VEGF 培养基的应用可以获得高纯度、具有良好生长特性的 BREC,是一种简便、效果稳定可靠的体外培养牛视网膜微血管内皮细胞的方法。

Purpose To probe a simple and reliable method for bovine retinal endothelial cells in vitro.Methods With the isolation of active retinal blood vessels,BREC were cultured in Dulbocco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum,heparin,and vascular endothelial growth factor(VEGF).BREC were identified by the speciality antibody to Von Willebrand factor and were studied by transelectromicroscope.Results VEGF can acerbate BREC proliferation.BREC got together around the microvessel fragments with the small-cyprinoid-like configuration.They stopped proliferating when they were contacted each other.The expression of Von Wllebrand factor was positive,and the purity was more than 95%.Conclusion BREC with high purity can be obtained by using conditioned DMEM supplemented with VEGF which is a simple and repeatable method.