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目的 建立胚胎及成人视网膜神经细胞体外培养系统 ,为视网膜神经细胞的基础研究与药物开发提供实验模型。 方法 10～ 13周龄胎儿及 2 0～ 40岁成人视网膜神经层用不同的酶进行消化 ,分散的细胞接种于预先经过包被的细胞培养盘内 ,加入或不加入表皮生长因子 (epidermal growth factor,EGF)、成纤维细胞生长因子 (fibroblast growth factor,FGF)、脑源性神经营养因子 (brainderived neu-rotrophic factor,BDNF)、神经营养素 - 4(neurotrophic- 4,NT- 4)等培养。通过 Brd U掺入、免疫组织化学及免疫荧光等方法检测培养细胞增殖并辨别细胞成分。 结果 胚胎及成人视网膜细胞可在体外连续培养10 0及 180 d以上。加入 EGF、FGF、BDNF或 NT- 4可明显促进胚胎与成人视网膜神经元存活 ,胎儿视网膜细胞增殖。与对照组相比 ,处理组神经元或神经节细胞的百分率较高。 结论 胚胎及成人视网膜神经细胞培养技术为视网膜神经细胞基础研究及药物开发提供了一种十分有价值的手段 ,加入外源性的生长因子可促进培养的视网膜神经细胞存活、增殖与分化。
Objective To establish an in vitro culture system of embryonic and adult retinal neurons and provide experimental models for basic research and drug development of retinal neurons. Methods The fetal retinal neurons of 10-13 weeks old and adults aged 20 to 40 were digested with different enzymes. The dispersed cells were inoculated into pre-coated cells and incubated with or without epidermal growth factor (EGF), fibroblast growth factor (FGF), brainderived neu-rotrophic factor (BDNF) and neurotrophic-4 (NT-4). Brd U incorporation, immunohistochemistry and immunofluorescence were used to detect the proliferation of cultured cells and identify the cell components. Results The embryonic and adult retinal cells can be continuously cultured in vitro for more than 10 0 and 180 days. Addition of EGF, FGF, BDNF or NT-4 significantly promoted embryonic and adult retinal neuronal survival and fetal retinal cell proliferation. Compared with the control group, the percentage of neurons or ganglion cells in the treated group was higher. Conclusion Embryonic and adult retinal nerve cell culture techniques provide a very valuable tool for basic research and drug development of retinal neurons. Addition of exogenous growth factors can promote the survival, proliferation and differentiation of cultured retinal neurons.