大鼠视网膜前体细胞的传代培养及体外分化诱导

Expansion culture and differential induction of rat retinal progenitor cells in vitro

目的了解表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)及睫状神经营养因子(CNTF)对其分化的影响。方法取孕18d的SD大鼠胚胎眼的视网膜,分别用悬浮法及贴壁法培养。传至第一代后转入分别含10ng/mlCNTF、20ng/mlEGF、10ng/mlbFGF、10%胎牛血清的DMEMF12中进行诱导分化,贴壁后2、3、4、6d分别行nestin、Vimentin、Opsin及GFAP的二步法免疫组织化学染色。结果大鼠的RPCs在悬浮时呈球状生长,nestin及Vimentin染色阳性,悬浮培养传至一代,贴壁培养传至五代,传代后细胞数量不断减少并可在体外分化为含视网膜光感受器细胞及胶质细胞的混合神经元。CNTF、bFGF及EGF不同程度地增加了光感受器细胞的比例。结论RPCs可在体外培养并传代。CNTF、bFGF及EGF参与视网膜发育的调节。

Objective Many researches have paid close attention on retinal transplantation in the treatment of retinal degenerative disease. Retinal progenitor cells （RPCs） have the ability to differentiate into multiple retinal cells,therefore to study the affecting factor of RPCs differentiation is critic for the rescue of degenerated retinal cells. This work was to investigate the differential potential of cultured rat retinal progenitor cells （RPCs） and its＇ affecting factors. Methods RPCs were isolated from retina of embryonic SD rat aged 18 days and cultured in suspension or attached condition. After expansion, RPCs were cultured in dulbecco＇ s modified eagle＇s medium （DMEM）, F12 containing 10 ng/ml ciliary neurotrophic factor（CNTF）, 20 ng/ml epidermal growth factor（EGF） ,10 ng/ml basic fibroblast growth factor （bFGF） and 10% fetal bovine serum （FBS）. After 2,3,4,6 days of cell attachment, cultured RPCs were identified by immunohistochemistry staining with nestin, Vimentin, Opsin and GFAP. Results Cultured RPCs showed a sphere shape in suspension and were stained 85% positive for nestin and Vimentin. The suspension RPCs were able to maintain in culture for the first generation. The attached RPCs were survival till the 5th generation,and the cell numbers was decreased with the passage. RPCs could differentiate into complex neurons including photoreceptor cells and glial cells and exhibited brown staining in cytoplasm for Opsin and GFAP. CNTF, bFGF and EGF could improve the ratio of photoreceptor cells differentiation,and FBS markedly increased the numbers of glial cells. Conclusion RPCs can be successfully cultured and proliferated in vitro. CNTF, bFGF, EGF and FBS participate in the regulation and control of retina development and differentiation.