摘要
目的建立无血清培养基培养纯化原代视网膜神经节细胞（RGCs）的方法，为研究青光眼等引起的RGCs损伤及防护提供理想的细胞模型。方法出生后1～3d的SD大鼠视网膜细胞悬液，分别经抗大鼠信号调节蛋白（CD172G）单克隆抗体筛除巨噬细胞、抗大鼠Thy-1单克隆抗体筛选RGCs的两步筛选法，得到纯化的RGCs。用加入B27、睫状神经营养因子等的无血清神经元专用神经基质（neurobasal）培养基进行培养，通过细胞形态学观察、Thy-1免疫荧光细胞化学染色、钙黄绿素-乙酰乙酸酯（AM）染色等方法观察原代培养的视网膜神经细胞及纯化培养的RGCs生长并进行鉴定。结果原代培养的视网膜细胞中约91％为神经细胞。纯化培养的RGCs接种后24h有突起长出；第4～8天可见细胞形态均一，细胞体较大，细胞突起较长；至第14天，仍有超过60％的细胞有活性。Thy-1的免疫荧光细胞染色结果显示RGCs纯度约为90％。钙黄绿素-AM染色存活的RGCs，结果显示RGCs细胞体较大，多数细胞有长度大于细胞体直径2倍的突起。结论该方法培养的RGCs大小均一、形态理想、纯度高，适宜研究各种因素引起的RGCs损伤及保护剂效果评价。
Objective To establish a purified model of rat retinal ganglion cells （RGCs） cultured by serum-free medium,and provide a good cell model to investigate the damage of RGCs in glaucoma,retinal ischemia, and degenerative retinopathy. Methods Two monoclonal antibodies,anti-rat SIRP （OX-41） against rat maerophage and antibody against rat Thy-1 （OX-7）,were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley （SD） rats by means of two-step filtration. Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor （CNTF） meeting the neuronal cell＇s special requirements. Photomicrographs illustration,immunfluorescence staining of Thy-1, calcein-acetoxymethyl ester （calcein-AM） fluorescence images were used to observe and identify cultured retinal ceils and purified RGCs. Results Among the primary cultured rat retinal cells,91% were retinal neurons. Protuberances of RGCs were seen after cultured for 24 hours. At the4th to 8th day,many cells had uniform configuration,large body,and long protuberances. At the 14th day,over 60% cells maintained viability. Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining,which stained the living cells only,showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size ,good configuration ,and high purity,which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2006年第3期200-203,共4页
Chinese Journal of Ocular Fundus Diseases
关键词
视网膜神经节细胞
培养基
无血清
荧光染色
疾病模型
动物
Retinal ganglion cells
Culture media,serum-free
Fluorescent dyes
Disease models ,animal
