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本文旨在建立低等灵长类动物树鼩(Tupaia belangeri)脑星形胶质细胞(astrocyte,AS)原代培养及纯化的技术,为利用新型实验动物树鼩进行研究工作而建立体外模型。将新生树鼩大脑皮质机械分离,置于4°C冰箱20min以损伤神经元,皮质组织块用胰蛋白酶消化后制成细胞悬液,分次贴壁去除成纤维细胞,培养的混合细胞在每次换液时用0.005%胰蛋白酶轻柔漂洗去除神经元。细胞长满培养瓶底面积约70%时,用0.025%胰酶溶液静置消化,至肉眼可见一层白色薄膜从瓶底脱落时终止消化,此白色薄膜即为AS层。AS传至第三代时,用抗胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)抗体进行免疫组织化学染色和免疫荧光染色鉴定。结果显示,本方法所得的树鼩脑AS的纯度可达98%以上。该结果提示,这种通过分次贴壁法结合差异消化的培养及纯化技术可获得高纯度的树鼩脑AS,为建立神经系统疾病新的体外细胞培养模型打下了基础。
The aim of this study was to establish a technique for primary culture and purification of astrocyte (AS) from Tupaia belangeri, and to establish an in vitro model for the study of a new experimental tree shrew. Neonatal tree cortex cerebral cortex mechanical separation, placed in 4 ° C refrigerator 20min to damage neurons, cortical tissue block digested with trypsin made of cell suspension, divided into adherent wall to remove fibroblasts, cultured mixed cells in each During fluid exchange, the neurons were gently rinsed with 0.005% trypsin to remove neurons. Cells covered with culture bottom area of about 70%, with 0.025% trypsin solution static digestion to the naked eye can see a white film from the bottom of the bottle to stop digestion, the white film is the AS layer. When AS was transmitted to the third generation, immunohistochemical staining and immunofluorescence staining were performed using anti-glial fibrillary acidic protein (GFAP) antibody. The results show that the purity of the tree 鼩 brain AS obtained by the method can reach more than 98%. The results suggest that this method of fractional adherent method combined with differential digestion of culture and purification technology to obtain high-purity tree 鼩 brain AS, to establish a new in vitro cell culture model of neurological diseases lay the foundation for cell culture.