牛脉络膜微血管内皮细胞纯化和体外培养的改良方法  

A modified method for purification and primary culture of bovine choroidal microvascular endothelial cells in vitro

作　　者：赵炜[1] 王雨生[1] 朱洁[1] 惠延年[1] 

机构地区：[1]第四军医大学西京医院眼科,全军眼科研究所,陕西省西安市710032

出　　处：《眼科新进展》2008年第3期161-165,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30371516,30672291);教育部留学回国人员科研启动基金资助(2004)~~

摘　　要：目的建立快速、简便地培养脉络膜微血管内皮细胞的方法,为脉络膜新生血管疾病的研究提供细胞模型。方法显微分离牛脉络膜微血管,采用1g·L-1胶原酶一步法消化,并采用免疫磁珠分选脉络膜微血管内皮细胞,使用内皮细胞培养基进行原代培养。利用光学和电子显微镜进行形态学观察,利用Von Willebrand因子免疫荧光染色及Dil-Ac-LDL吞噬实验进行细胞学鉴定,利用管腔形成实验观察内皮细胞形成管腔能力。结果采用本研究改良的方法可以获得纯度高达95%的脉络膜微血管内皮细胞,外观呈细长形,融合后呈典型铺路石样外观。电子显微镜观察可见胞浆内靠近核膜外的Weibel-Palade小体(棒状小体);免疫荧光显示Von Willebrand因子表达阳性,Dil-Ac-LDL吞噬实验阳性;在凝胶中可以形成明显管腔结构。结论本研究改良的方法成功地分离并纯化了牛脉络膜微血管内皮细胞,可在短期内快速简便地获得足够数量的内皮细胞。Objective To establish a rapid and convenient method for purification and primary culture of bovine choroidal microvascular endothelial cells（CMEC） in vitro and to provide a cell model for investigating choroidal neovascularization （CNV） diseases.Methods Bovine choroidal microvascular vessels were isolated from choroidal tissues by microdissection and digested with 1 g·L-1 collagenase.CMEC were purified selectively by immunomagnetic beads and cultured with endothelial culture medium.The morphological characteristics of CMEC were observed by light and electron microscopy and identified by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis.The ability of CMEC forming tubes was examined with tube formation experiment.Results There were 95% of the cultured cells which were CMEC affirmed by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis.The cultured CMEC were slender.After confluence,the cells had cobble-stone appearance.Weibel-Palade body could be found near nuclear membrane by electron microscopy.The staining of Von Willebrand factor was positive and the cells could phagocytose the Dil-Ac-LDL.Conclusion Bovine CMEC can be successfully isolated and purified with this modified method by which enough CMEC can be got easily for CNV study.

关 键 词：脉络膜微血管内皮细胞 牛 细胞培养 

分 类 号：R773.4[医药卫生—眼科]