成人视乳头星形胶质细胞的纯化培养及鉴定

Purification and characterization of adult optic nerve head astrocytes

目的探讨稳定的成人视乳头星形胶质细胞原代纯化培养方法及其理想细胞模型。方法实验研究。解剖显微镜下分离出人眼视乳头筛板组织，将其剪切成4—6个小组织块，放置于含有DMEM／F12培养基的培养皿中培养。8～10周后去除组织块，以0．25％胰蛋白酶消化细胞，改用星形胶质细胞选择性培养基，经两次传代后，通过细胞形态学和胶质细胞原纤维酸性蛋白、神经细胞黏附分子免疫荧光染色，观察纯化培养的星形胶质细胞生长情况并进行细胞鉴定。结果组织块接种2～3周后，有细胞相继从其边缘爬出，迅速向周围分裂生长，其移行过程明显。胰酶消化后细胞的形态多呈扁平星形或多角形，也可见长梭形细胞。改用星形胶质细胞选择性培养基培养并传代后，细胞几乎均呈扁平星形，并表达胶质细胞原纤维酸性蛋白和神经细胞黏附分子。结论精确分离筛板组织并将小组织块定位于培养液边缘（培养液并未完全覆盖皿底），有利于组织块贴壁生长，是获取人原代星形胶质细胞的前提条件；星形胶质细胞选择性培养基是获得原代纯化星形胶质细胞的简便有效方法。

Objective To establish a method of purifying and characterizing adult astrocytes from optic nerve head （ONH）. Methods Experimental study. The lamina cribiosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting ceils with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. Results After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a fiat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. Conclusions Cultured human ONH astrocytes can be obtained by precisely separating lamina cribiosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.