摘要
目的 探讨牛视网膜微血管内皮细胞 (BREC)和周细胞 (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、10 0 μg/ ml肝素的 Dulbecco改良 Eagle培养基(DMEM)和含 2 0 %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 (Dil- Ac- L DL )吞噬情况 ,应用 Von Willebrand因子抗体通过免疫组织化学方法鉴定BREC,并通过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 ;BRP多散布在距血管碎片稍远处 ,形状不规则 ,呈无接触性抑制生长。BREC可吞噬 Dil- Ac- L DL,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。
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 100 μg/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 immunohistochemical 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 respectively in BREC, while were negative and positive respectively in BRP. Conclusion BREC and BRP with high purity can be obtained by using selective culture and coated-dishes respectively which are simple and repeatable methods.
出处
《中华眼底病杂志》
CAS
CSCD
2004年第1期23-26,共4页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金资助项目 (39970 783)
上海市卫生系统优秀学科带头人培养计划资助项目 (97BR0 31 0 )
