牛视网膜微血管内皮细胞和周细胞的体外培养

目的 探讨牛视网膜微血管内皮细胞（ bovine retinal endothelial cells, BREC ）和周细胞（bovine retinal pericytes, BRP）的体外选择性培养方法。 方法 结合视网膜微血管的消化分离，采用含10％人血清、100 μg/ml 肝素（Heparin）的Dulbecco改良Eagle培养基(Dulbecco′s modified Eagle′s medium, DMEM)和含20％胎牛血清的DMEM培养基分别选择性培养BREC和BRP。通过荧光显微镜观察乙酰化低密度脂蛋白(acetylated low density lipoprotein, Dil-Ac-LDL)吞噬情况并应用Von Willebrand因子抗体通过免疫组织化学方法鉴定BREC；以及α-平滑肌肌动蛋白抗体免疫组织化学鉴定BRP。 结果 通过选择性培养获得的BREC和BRP的纯度达到98％以上，并能连续传代。BREC早期似小鲤鱼状聚集在微血管碎片周围，呈片状鹅卵石样单层生长，存在接触性抑制；周细胞多散布在距血管碎片稍远处，形状不规则，呈无接触性抑制的生长。BREC可吞噬Dil-Ac-LDL，细胞浆内有荧光表达 ；消化传代后BREC中Von Willebrand因子表达阳性，而α-平滑肌肌动蛋白表达阴性；BRP的α-平滑肌肌动蛋白表达阳性，而Von Willebrand因子表达阴性。 结论 选择性培养基的应用和培养皿的处理，可分别获得较高纯度的BREC和BRP，简单且具有良好的重复性，无需额外步骤来去除BREC中混杂的BRP。 （中华眼底病杂志，2004，20：23-26）

Primary culture of bovine retinal capillary endothelial cells and pericytes in vitro

Objective To probe a selective cultural method for bovine retinal endothelial cells (BREC) and pericytes (BRP) in vitro.Methods With the isolation of active retinal blood vessels, BREC were cultured in a fibronectin coated substrate and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% human serum and 100μg/ml heparin, while homogeneous cultures of retinal pericytes were obtained when isolated microvessels were seeded to uncoated dishes and grown in DMEM supplemented with 20% fetal bovine serum. BREC were identified by acetylated-low density lipoprotein (Dil-Ac-LDL) incorporation and immunohistochemical method of Von Willebrand factor, while BRP were identified by the immunohist ochemical method of α-isoform of smooth-muscle actin. Results The purity of selectively cultured BREC and BRP was more than 98%, being reproducible. BREC got together around the microvessel fragments with the small-cyprinoid-like configuration at first，and could phagocytize Dil-Ac-LDL with the expression of fluorescence in cytoplasm. The expressions of Von Wllebrand factor and α-isoform of smooth-muscle actin were positive and negative in BREC respectively, while were negative and positive in BRP respectively.Conclusion BREC and BRP with high purity can be obtained by using selective culture and coated-dishes respectively which are simple and repeatable methods. (Chin J Ocul Fundus Dis，2004，20：23-26)