大鼠视盘筛板星形胶质细胞的培养纯化及鉴定

Purification and characterization of rat optic nerve head astrocytes

目的探讨大鼠视盘筛板部位原代星形胶质细胞体外分离纯化培养方法。方法在解剖显微镜下分离成年大鼠视盘及其后一小段视神经,切块后放入含DMEM/F-12培养瓶中进行原代培养。经2.5 g·L-1胰蛋白酶消化、星形胶质细胞选择性培养基纯化,传代2次后,用胶质细胞标志性抗体胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经元细胞黏附分子(neural cell adhesion molecule,NCAM)和波形蛋白(Vimentin)进行免疫荧光化学染色;证实为星形胶质细胞后,用表皮生长因子刺激星形胶质细胞24 h,观察其形态变化并用Western blot法检测培养细胞GAFP、NCAM和Vimentin蛋白的表达情况。结果组织块贴壁后10 d左右,细胞开始爬出,且形态各异。经星形胶质细胞选择性培养基纯化后,细胞形态多呈星形或不规则形,胞质丰富;免疫荧光化学染色显示GFAP、NCAM、Vimentin蛋白染色阳性,阳性细胞率达90%以上;表皮生长因子刺激后,细胞增生,形状发生改变,且与空白对照组相比,刺激后GAFP、Vimentin和NCAM蛋白表达增加,差异均有统计学意义(均为P<0.05)。结论本方法可在体外培养并获得纯度较高的大鼠视盘筛板部位的原代星形胶质细胞。

Objective T o develop a set of methods for the culture and purification of astrocytes grow n from explanted rat optic disc. Methods Rat optic disc and optic nerve w ere separated w ith anatomic microscopy and the blocks w ere cultivated primarily in culture flask w ith the medium containing DM EM/F-12. After digested by 2. 5 g·L-1 trypsin,astrocytes w ere purified on the selective medium and passaged for tw o generations.T hen cells w ere identified by immunofluorescent staining of glial fibrillary acidic protein( GFAP),neural cell adhesion molecule( NC AM) and Vimentin. Next,epidermal grow th factor( EGF) w as used to stimulate the purified cells for 24 h to observe their morphologic alteration. Finally,W estern blot w as used to detect the expression of GAFP,NC AM and Vimentin in the cells. Results W hen the tissue blocks w ere attached to the w all for 10 days,cells began to craw l 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 above90%. After stimulated by EGF,cells proliferated and the morphology changed; meanwhile,t here 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.