不同时期视网膜前体细胞的培养

CULTIVATION OF RETINAL PROGENITOR CELLS OF DIFFERENT AGES

目的研究不同时期视网膜前体细胞的培养。方法分离E14和E18 SD大鼠的视网膜前体细胞,用改进DMEM/F12无血清培养基悬浮培养并利用体外诱导分化、免疫细胞化学、扫描电镜和透射电镜等方法对细胞进行鉴定。结果视网膜前体细胞在DMEM/F12无血清培养基中悬浮成球,贴壁后,细胞逐渐从细胞球向周围迁移分化。扫描电镜可见细胞球和分化细胞的形态,透射电镜可见细胞球和分化细胞的超微结构。免疫细胞化学显示,悬浮培养的细胞球中大部分细胞表达神经外胚层源性干细胞标志物nestin和细胞分裂增殖标志物BrdU。贴壁后,视网膜前体细胞能分化为多种视网膜细胞类型,包括Thy1.1阳性的视网膜神经节细胞。E14和E18视网膜神经节细胞的百分比分别为16.91%±4.05%和4.65%±1.88%,两组比较,差异有统计学意义(t=15.04,P<0.001)。结论改进DMEM/F12无血清培养基可成功培养视网膜前体细胞,培养的细胞具有无限增殖和多向分化潜能。早期视网膜前体细胞向视网膜神经节细胞分化的百分比较高。

Objective To study the cultivation of retinal progenitor cells of different ages. Methods Retinal progenitor cells of El4 and E18 SD rats were isolated, cultivated in suspension in modified DMEM/F12 serum-free medium and then differentiation was induced in vitro. Cells were observed under phase-contrast microscopy daily and identified by immunocytochemistry, scanning electron microscopy and transmission electron microscopy. Results Cultivated in DMEM/F12 serum free medium, retinal progenitor cells formed cell spheres. After being plated, isolated cells migrated outwards and differentiated. Scanning electron microscopy demonstrated the morphology of the cell spheres and differentiated cells. Transmission electron microscopy demonstrated that there were stem cell-like cells in cell spheres and neuron- and glia-like cells after the plating. Immunocytochemistry demonstrated that most cells in spheres expressed neural stem cell marker nestin and cell division marker BrdU. After the plating, retinal progenitor cells could be induced to differentiate into various retinal cells, including Thyl. 1- positive retinal ganglion cells. The percentage of retinal ganglion cells was 16.91% ± 4.05% at El4 and 4.65% ：t： 1.88% at E18 .The differences were statistically significant（ t = 15.04, P 〈 0.001 ）. Conclusion Retinal progenitor cells can be cultivated successfully in modified DMEM/F12 serum-free medium. The cultivated retinal progenitor cells have the potential to proliferate freely and multi-differentiate. Early retinal progenitor cells are inclined to differentiate into retinal ganglion cells.