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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 method to culture and purify primary retinal ganglion cells (RGCs) in serum-free medium, and to provide an ideal cell model for studying the damage and protection of RGCs caused by glaucoma. Methods 1 ~ 3d SD rat retinal cell suspension was used to screen macrophages by anti-rat signaling protein (CD172G) monoclonal antibody and anti-rat Thy-1 monoclonal antibody screening RGCs by two-step screening Method to obtain purified RGCs. The cells were cultured in neurobasal medium supplemented with B27, ciliary neurotrophic factor and other serum-free neurons. Thy-1 immunofluorescence cytochemistry, Calcein-acetoacetate (AM) ) Staining and other methods to observe the primary culture of retinal nerve cells and purified culture RGCs growth and identification. Results About 91% of primary cultured retinal cells were nerve cells. After cultured for 24h, the cultured cells grew prominently. On the 4th to 8th day, the cell morphology was uniform, the cell body was larger and the cell protuberance was longer. By the 14th day, more than 60% cells were still active. Immunofluorescence staining of Thy-1 showed that the purity of RGCs was about 90%. Calcein-AM staining of surviving RGCs showed that RGCs had large cell bodies, and most of the cells had protrusions twice as long as the cell body’s diameter. CONCLUSION RGCs cultured in this method are uniform in size, ideal in morphology and high in purity, and are suitable for studying RGCs injury caused by various factors and evaluation of the effect of protective agents.