摘要
取生后１周以内ＳＤ大鼠大脑皮质，用胰蛋白酶消化结合机械吹打使细胞分散，制成较大量的初细胞悬液。将初细胞悬液先行粘附处理３０ｍｉｎ以排除其中的成纤维细胞，再制成较小量的次细胞悬液。将之接种到预先涂有鼠尾胶原的培养瓶中，令其贴壁３～５ｈ，细胞密度约１×１０５个／ｃｍ２。补加培养液，继续培养。培养液为含２０％小牛血清的ＤＭＥＭ／Ｆ１２混合培养液。俟细胞铺满瓶底后传代。届时细胞悬液亦行３０ｍｉｎ的粘附处理。以０．５×１０５个／ｃｍ２的细胞密度种植于培养瓶中。传代后先培养４８ｈ；换成无血清培养液再培养４８ｈ并收集条件培养液。对部分传代细胞进行胶质原纤维酸性蛋白免疫细胞化学杂色鉴定，证明传代后的细胞中星形胶质细胞比例达９４．７１％。取条件培养液及单纯ＤＭＥＭ／Ｆ１２培养液各１份，分别与２份有血清培养液混合，制成实验组与对照组两种培养液，以悬滴法培养鸡胚背根节。４８ｈ后，比较两组背根节神经突起的平均长度。经统计学分析揭示，实验组背根节神经突起平均长度显著大于对照组者，说明星形胶质细胞具有明显的促神经突起生长作用。
Astrocyte-enriched culture from postnatal rat(within 1-week)cerebral cortex was prepared. Cerebral tissue was dissected and dispersed by trypsinization as well as mechsnical method to make an initial cell suspension of a volume of shout 20ml.This suspension was seeded in a 250 ml culture flask and incubated for 30 minutes to eliminate fibroblasts based on their preferentially adhesive properties compared with the other cell types.The treated suspension was then filtered through a 75μm-pore stainless steel sieve.The filtrate was centrifuged and the cell pellet resuspended into a second cell suspension of about 2 ml. This suspension was plated at a density of 1×10￣5 cells/cm￣2 in a 250 ml culture flask coated with rat-tail collagen and kept in a 37℃,5 % CO_2 incubator for 3～5 hours so as to attach more quickly and firmly to growth substratum. Then,nutritional fluid which contained 20% newborn calf serum and 80% DMEM/F12 (DMEM:F12 1:1)was supplemented to the flask and cultured again.This was the primary culture of rat cerebral cells.Nine～14 days later,when cells were at confluency, the primary culture was passaged.After another adhesion treatment, the dispersed cells were replated at a density of 0.5×10￣5 cells/cm￣2 in a 75 ml flask coated with rat-tail collagen.Passaged cells were cultured continuously with the above-mentioned nutritional fluid for 48 hours,and then changed with a serum-free DMEM/F12 medium. FOrty-eight hours later, conditioned medium was collected.Some passaged cells were immunocytochemically stained with anti- gilal fibrillary acidic protein(GFAP)antibody to identify astrocytes which made 94. 71 percent of total cells.In order to study the neuriteoutgrowth promoting effect of the cultured astrocytes,Hanging-drop culture technique was employed to culture chicken dorsal root ganglia at embryonic day 9. Culture medium consisted of 1/3 astrocyte conditioned medium and 2/3 DMEM/F12medium supplemented with 20 % calf serum,and the control group medium consisted of 1/3 DMEM/F12 medium instead of conditioned medium and 2/3 serum.After 48 hours culture,the mean neurite length of two groups were measured. Statistical analysis demonstrated that the mean neurite length of the experimental group was significantly greater than that of the control.This result suggest that astrocyte may have neurite-outgrowth promoting effect.
出处
《神经解剖学杂志》
CSCD
北大核心
1996年第2期151-155,共5页
Chinese Journal of Neuroanatomy
关键词
大脑皮质
星形胶质细胞
细胞培养
astrocyte
cell culture
neurite-outgrowth
cerebral cortex
rat
