胚胎与成人视网膜神经细胞培养

The cultivation of fetal and adult human retinal cells

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 a culture system in vitro of fetal and adult human retinal neural cells provide a model for the basic research of retinal neural cells and the medicinal exploitation. Methods Fetal human retinas(10~13 weeks after conception) and adult human retinas(20~40 years old) were dissected, dissociated, and put into culture plate which was coated with polylysine or rat tail gel. Specific growth factor EGF?FGF? BDNF or NT-4 were added to the culture medium. BrdU incorporation, Tunnel assessment and immuno-histochemistry and immuno-fluorescent staining were applied to determine cells proliferation, apoptosis and identify the component of cultured cells. Results Fetal human retinal cells and adult human retinal cells survived for up to 100 and 180 days in vitro. The addition of EGF?FGF? BDNF or NT-4 promoted the survival of both fetal and adult retinal neurons and stimultated proliferation of fetal retinal cells. The neurons or the rate of ganglion cells was observed with higher percentage in the group with growth factor adding than the group without. Conclusion Fetal and adult human retinal cells can be maintained in vitro and the fetal cells also can be expanded, which are helpful to generate retinal neurons for basic research and drug exploitation. The exogenous growth factors added to the culture medium can promote survival, proliferation and differentiation of retinal cells in culture.