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目的探讨体外培养海马细胞的生物学特性及缺氧缺血模型建立的方法。方法无菌手术剥离、收集24h新生大鼠海马,胰酶消化后行Neurobasal无血清培养基培养。相差显微镜下观察细胞形态,采用CCK-8法检测细胞活力,神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP-2)和胶质纤维酸性蛋白(GFAP)免疫荧光染色鉴定海马细胞,氧糖剥夺法建立体外缺氧缺血模型。结果细胞接种后,细胞开始呈对数生长趋势,7d后呈现平台期。NSE和MAP-2在细胞中均呈阳性表达,而GFAP为阴性表达。氧糖剥夺严重损伤海马细胞;随着剥夺时间的延长,细胞活性呈进行性下降。结论体外培养可获得生长旺盛的海马细胞;氧糖剥夺实验可作为体外缺氧缺血模型建立的方法。
Objective To investigate the biological characteristics of cultured hippocampal cells and the establishment of hypoxic-ischemic model. Methods Aseptic surgery was performed and the hippocampus of neonatal rats were collected for 24h. After trypsinization, the cells were cultured in serum-free medium of Neurobasal. Cell morphology was observed under a phase contrast microscope. Cell viability, neuron-specific enolase (NSE), MAP-2 and GFAP immunofluorescence staining were used to detect cell viability by CCK-8 assay Hippocampal cells, oxygen deprivation method to establish an in vitro hypoxic-ischemic model. Results After cell inoculation, the cells began to show a logarithmic growth trend, and showed a plateau after 7 days. Both NSE and MAP-2 were positive in cells and negative in GFAP. Oxygen-deprived severely damaged hippocampal cells; With the extension of deprivation of time, cell activity decreased progressively. Conclusion In vitro culture of hippocampal cells can be vigorous growth; oxygen-glucose deprivation experiments can be used as a model of hypoxic-ischemic in vitro established.