ABSTRACT
Objective To develop a set of methods for the culture and purification of astrocytes grown from explanted rat optic disc.Methods Rat optic disc and optic nerve were separated with anatomic microscopy and the blocks were cultivated primarily in culture flask with the medium containing DMEM/F-12.After digested by 2.5 g · L-1 trypsin,astrocytes were purified on the selective medium and passaged for two generations.Then cells were identified by immunofluorescent staining of glial fibrillary acidic protein (GFAP),neural cell adhesion molecule (NCAM) and Vimentin.Next,epidermal growth factor (EGF) was used to stimulate the purified cells for 24 h to observe their morphologic alteration.Finally,Western blot was used to detect the expression of GAFP,NCAM and Vimentin in the cells.Results When the tissue blocks were attached to the wall for 10 days,cells began to crawl out of the tissues and had different morphologies.After purified by the selective culture medium,satellite or anomalistic cells,possessing abundant cytoplasm,began to take a leading place.Immunofluorescent staining showed GFAP,NCAM and Vimentin were stained positively,and the purity of positive cells was above 90％.After stimulated by EGF,cells proliferated and the morphology changed;meanwhile,there was an increasing expression of GFAP,NCAM and Vimentin after stimulated by EGF when compared with the blank control group,and the difference was statistically significant (P ＜ 0.05).Conclusion Highly-purified astrocytes can be achieved by the culture of explanted lamina cribiosa and optic disc of rats in vitro.