新生大鼠视网膜神经元及节细胞体外短期培养方法  

Short period culture of retinal neurons and ganglion cells of neonatal rat in vitro

作　　者：白海青[1] 王竫华[1] 王大博[1] 李树宁[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：《眼科新进展》2004年第2期107-110,共4页Recent Advances in Ophthalmology

摘　　要：目的　建立新生大鼠视网膜神经元原代培养体系 ,观察视网膜神经元和神经节细胞 (retinalganglioncells,RGC)在体外短期培养的生长特点。方法　将新生大鼠视网膜细胞悬液接种于预先用鼠尾胶原包被的 96孔细胞培养板中 ,按 4 0 0× 10 3 ·cm-2 (高密度 )及 2 0 0× 10 3 ·cm-2 (低密度 ) 2种密度接种 ,抑制非神经元生长 ,MTT比色法评价细胞活力 ;上述细胞悬液按高密度接种于 2 4孔细胞培养板中 ,进行HE染色和Thy1单克隆抗体的免疫化学染色 ,测量视网膜神经元和RGC轴突的长度。结果　视网膜神经细胞有聚集成簇生长的倾向 ,细胞活力在高密度培养时明显高于低密度培养 ;第 3天细胞活力最高 ;视网膜神经元在体外形态多样 ,最初 2d轴突生长速度最快 ,超过 12 .0 μm·d-1;大多数RGC从第 1天就再生出 2个或 2个以上的突起 ,第 1天的平均轴突长度为 12 .5 μm·d-1,从第 2天起保持在8 0 μm·d-1。第 4天的平均轴突长度为 2 9.4 5 μm。结论　以鼠尾胶原为支持物 ,经过酶消化法得到的新生大鼠视网膜神经元 ,包括RGC ,能够在短期内表现出较高的细胞活力并再生出较长的轴突 ;高密度培养 ( 4 0 0× 10 3 ·cm-2 )更有利于视网膜神经元在体外生长存活。Objective To observe the growth characters of retinal neurons and retinal ganglion cells (RGCs) by setting up retinal neurons in vitro culture system of neonatal rats.Methods Monocell suspend from neonatal rats retina was seeded into 96 hole culture plates pre coated by murine tail collagen. Cells densities were 400×10 3·cm -2 and 200×10 3·cm -2 respectively. The proliferation of non neurons was inhibited. MTT chromatometry was applied to evaluate the viability of neurons at day 1,2,3 and 4.The above retinal cells suspend was also planted in 24 hole culture plates with cover glasses inside pre treated by murine tail collagen.And the cover glasses with retinal neurons on it were fixed at day 1,2, 3 and 4 in turn so as to carry on hematoxylin eosin (HE) stain and anti Thy 1 monoclonal antibody immunocytochemistry. Then the axon length of retinal neurons and RGCs were measured from the 1st to 4th day.Results Retinal neurons had the tendency to grow in clusters. Neurons viability at high density culture was significantly higher than that at low density and it reached the peak at the 3rd day, then declined. Retinal neurons showed various morphologic shapes when cultured in vitro .Retinal neurons got their highest axon growth speed at the first 2 days,which was faster than 12.0 μm·d -1 , and slowed down from the 3rd day. RGCs displayed large and round nuclei and relatively less plasma.Most RGCs regrowed 2 or more processes at the 1st day. The mean axon extending speed was 12.5μm·d -1 at the 1st day, then maintains 8.0 μm·d -1 from the 2nd day. And their mean axons length reached the longest at the 4th day, which was about 29.45μm.Conclusion Retinal neurons including RGCs cultured from enzymatically digested retina supported by murine tail collagen show high viability and regrow longer axons in short period. High density culture (400×10 3·cm -2 ) is more benefited to the survival of retinal neurons in vitro.

关 键 词：大鼠 视网膜神经元 视网膜神经节细胞 存活力 轴突 

分 类 号：R771[医药卫生—眼科]