兔胚胎视网膜前体细胞体外培养纯化方法探究

Research on the isolation, culture and identification of rabbit retinal progenitor cells in vitro

目的探讨新西兰白兔的胚胎视网膜前体细胞的培养、增殖与纯化方法,并对其性质及体外分化进行鉴定。方法来源于19d、20d及26d不同孕龄的兔胚胎,用内路法取出含视网膜前体细胞的组织,采用胰酶/EDTA消化,利用组织中不同类型细胞对消化液不同的反应,去除混入的间质细胞等成分,达到逐步分离纯化胚胎视网膜前体细胞的目的。对分离所得的细胞,利用倒置相差显微镜观察其生长5情况,同时对相关类型细胞行免疫细胞化学染色,以鉴定视网膜前体细胞。结果从兔胚胎视网膜神经部分离出的原代视网膜前体细胞具有明显的生物学特性:所培养细胞可形成悬浮生长的细胞团,刚贴壁的细胞成簇状生长,这些细胞用免疫细胞化学染色神经上皮干细胞蛋白(Nestin)表达呈阳性。经传代几代后细胞生长趋同,细胞生长呈长梭形,逐渐有细胞分化为多种视网膜细胞类型,包括视网膜神经节细胞等,同时Nestin表达下调。结论不同时期、不同培养条件下的视网膜前体细胞均具有相同的形态,这种利用酶消化时间的不同能够分离纯化视网膜前体细胞,为将来研究此类细胞的性质及干细胞的相关研究打下良好的细胞生物学基础。

Objective To study the methodology for culture, proliferation and purification of retinal progenitor cells （RPCs）, and to identify and characterize those cells and their differentiation in vitro. Methods Retinal progenitor cells were extracted from 19-day, 20-day, and 26-day rabbit embryos from different lineages and digested with trypsin/EDTA. Mesenchymal cells are separated from RPCs based on the difference in the reaction to digestion. The growth of RPCs was observed with an inverted contrast phase microscope, and immunocytochemistry was used to characterize and identify harvested cells. Results Primary RPCs extracted from the neural retina demonstrate certain biological characteristics： the cell population could form neurospheres and younger adherent cells expanded in clusters. These ceils were positive for Nestin, a marker for neural stem cells. After several passages, they changed into a spindle shape and started to differentiate into several types of mature retinal cells, including retinal ganglion cells. In addition, the expression of Nestin was down regulated in the cells. Conclusion RPCs from embryos at different ages and under different culture conditions have identical morphology. They can be purified on the basis of the diverse reactions of the different types of cells to Trypsin.