鼠视网膜血管内皮细胞体外培养的改良及鉴定

背景视网膜血管内皮细胞（RVECs）的体外培养是进行视网膜血管性疾病研究的基础，人眼球供体的缺乏是限制人血管内皮细胞培养的主要原因，与人高度同源的大鼠血管内皮细胞的培养方法已经建立，但其分离方法对细胞损伤较大，影响细胞的培养效率。目的建立和改良大鼠RVECs的培养方法。方法选取5只SD大鼠眼球，获得视网膜，用组织块培养法，将大鼠视网膜用质量分数0．1％胶原酶消化，并加入质量分数20％胎牛血清、血管内皮生长因子（VEGF）、血管内皮细胞生长添加物（ECGS），用血管内皮细胞培养基中和后充分吹打，制备细胞及组织悬液，接种于人纤维粘连蛋白（FN）包被的培养瓶中。用Ⅷ因子相关抗体鉴定培养的内皮细胞。结果组织块法消化培养2d可见细胞从组织块游出，培养4d可伸出触角呈梭形，5～6d可融合生长，培养14d后的细胞呈单层生长，培养的细胞Ⅷ因子相关抗体染色阳性。传代培养的细胞接种2h可重新贴壁，恢复融合生长状态。结论视网膜组织块培养并用胶原酶消化可获得活性较强的细胞和碎片，再用高选择性的血管内皮细胞培养基和FN促贴壁进行选择性培养，可获得纯度较高的RVECs。

Modification of culture method of retinal vascular endothelial cells in vitro

Background The in vitro culture of retinal vascular endothelial cells is the foundation of experimental study of retinal vascular disease. Shortage of human donor eyeballs is a main limiting for the laboratory work. The culture method of rat-derived vascular endothelial cells has been established. However, this method is not enough effective because of severer cellullar injury. Objective Present study was to establish a simple and high effective method for the culture of vascular endothelial cells in vitro. Methods The retinas from 5 SPF SD rats was digested by 0. 1% collagenase and cultured with explant culture method. 20% fetal bovine serum, vascular endothelial growth factor ( VEGF) , insulin-transferrin-selenium( ITS) were composed into the endothelial cell culture medium, and enough blowing was performed to get the cells and fragments from retinal tissue. The cellular suspension was prepared and cultured consequently on human fibronectin-coated culture flasks. Cultured vascular endothelial cells were identified by anti-von Willebrand staining factor. Results The cells emerged from the tissue mass,and cells and some tissue fragments attached to the wall after 24 hours of seeding. The cells grew to show the fusiform in 4 days and merged together in 5 to 6 days,and a cell monolayer was seen in the 14th day after culture. The endothelial cells showed the positive response for von Willebrand factor. After passage, the merging-growth statue of the cells was regained in 2 hours after culture. Conclusion Use of retinal pieces and collagenase-digestion can get the vascular endothelial cells with better activity in vitro. The culture method based on highly selective endothelial cell culture medium associated to FN adhesion-promoting is helpful for gaining the purified of endothelial cells.