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Purified retinal ganglion cells cultured in serum-free neurobasal medium
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
- Issue:
- 3
- DOI:
- 10.3760/j.issn:1005-1015.2006.03.015
- Key Word:
- 视网膜神经节细胞;培养基,无血清;荧光染色;疾病模型,动物
Abstract： 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1～3 d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24 h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.
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