新生大鼠视网膜神经元的原代培养与观察  

Primary culture of retinal neuron cells of neonatal rat

作　　者：刘夫玲[1] 牛膺筠[1] 王建波[1] 曲虹[1] 张蕾[1] 

机构地区：[1]青岛大学医学院附属医院眼科,山东青岛266003

出　　处：《山东大学学报（医学版）》2006年第7期684-688,共5页Journal of Shandong University:Health Sciences

基　　金：山东省科技攻关项目资助课题(2004GGB14112)

摘　　要：目的：建立新生大鼠视网膜神经元体外培养的有效方法。方法：取出生后1～3d的Wistar大鼠视网膜，胰蛋白酶＋依地酸（EDTA）消化制成单细胞悬液，接种于置有包被多聚赖氨酸（poly-L-lysine）玻片的24孔培养板中培养，利用相差显微镜对培养的细胞进行动态观察；并以免疫细胞化学技术对视网膜神经元进行染色。结果：在多聚赖氨酸上培养的视网膜神经元生长良好，部分细胞伸出突起，且有些突起相互连接。免疫细胞化学染色显示，培养的细胞大多数抗神经丝蛋白（NF）抗体反应阳性。胰蛋白酶＋EDTA联合消化能使视网膜细胞很好得分离，获得视网膜单细胞悬液。结论：酶消化法原代培养视网膜神经元有较好的视网膜神经元生长率；胰蛋白酶＋EDTA联合消化较单独用胰蛋白酶消化效果好。Objective： To establish a method of culture of retinal neuron cells in vitro. Methods： Cell suspension was made from retina of postnatal 1 ～ 3 day Wistar rat by using trypsin and ethylene diamion tet raacetic acid （EDTA） digestion, and then it was planted to cover slips covered with Poly-L-lysine in 24 well plates. The growth regularity of cells in vitro was observed under the phase contrast microscope, and the cells were identified by using immunohistochemistry, and then the NF-positive ones were counted under the light microscope. Results： Retinal neuron cells cultivated in Poly-L-lysine grew very well and some possessed axons, of which some were connected with each other. Most cells were NF -positive detected by immuniohistochemistry. Trypsin and ethylene diamion tet raacetic acid （EDTA） had salutary effect for making retina tissues into cell suspension. Conclusion： Cultivation of retinal neuron cells using enzyme digestion well develops in vitro. Trypsin combined with ethylene diamion tet raacetic acid （EDTA） is better than trypsin alone for digestion of epithelium in neurosensory retina.

关 键 词：视网膜 神经元 细胞培养 神经丝蛋白 

分 类 号：R774[医药卫生—眼科]