人视网膜前体细胞的体外培养、纯化和诱导分化

Culture,purification and differentiation in vitro of human retinal progenitors

目的 体外培养和纯化人视网膜前体细胞,研究其分化的诱导因素.方法 培养3～5个月人胚胎视网膜前体细胞,鉴定其神经干细胞特性.流式细胞术比较其传代前后的含量.观察不同培养条件对其分化的诱导.结果 人视网膜前体细胞能分裂增殖,形成原代和子代神经球,表达Nestin,分化后表达神经烯醇化酶、胶质纤维酸性蛋白、蛋白激酶C和神经突触素.传代后Nestin表达量[（39.3±18.9）%]显著高于原代[（28.7±17.7）%]（P＜0.05）.视网膜组织片上清液诱导后神经烯醇化酶表达量[（69.3±6.5）%]高于自发分化者[（59.5±7.6）%]（P＜0.05）.视紫红质表达量[（7.5±2.9）%]则低于自发分化者[（13.2±3.2）%]（P＜0.05）.组织共培养后,同一区域内细胞集中向特定细胞分化.结论 体外培养的人视网膜前体细胞能自我纯化,不同诱导形式使其分化发生偏差.

Objective To study the expansion and purification of human retinal progenitors cultured in vitro. Methods Human embryonic retinal progenitor cells （RPCs） were cultured. The characteristics of neural stem cell were observed. The expression of Nestin was examined flow cytometry （FCM） for the comparsion between the pri- mary and secondary passages. The differentiation of RPCs had been observed after that they had been cultured in different conditions. Results Human RPCs had proliferated and formed cell clusters with a shape of neurosphere. After digestion, the scattered progenitors formed secondary neurosphere-like cell clusters. The cells in those clusters expressed Nestin and were negative for the mature retinal cell proteins. After spontaneous differentiation, the retinal progenitors expressed special proteins of mature retinal cells, including neuron specific enolase（ NSE）, glial fibrillary acidic protein （GFAP）, protein kinase C （PKC） and Syntaxin-1. The expression of Nestin in the secondary culture passage was （39.3 ±18.9）%, which was significantly higher than that of the primary passage [ （28.7 ±17.7 ） % ] （ P 〈 0.05 ）. After being cultured in the medium with supernatant fluid of retina explant culture medium, the RPCs differentiated. The expression of NSE [ （69.3 ± 6.5 ） % ] was significantly higher than that of differentiated RPCs were homogeneously become special mature cells in the same area. Conclusion manifested characteristics of neural stem cells and purified spontaneously in selective culture medium. The differeniation of RPCs can be modulated by special conditions.