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目的:创建理想的纯化嗅鞘细胞(OECs)的方法,并观察不同时间段OECs上清液中神经营养因子的含量,为选择OECs移植的最佳时机提供理论依据。方法:采用差速贴壁法结合免疫亲和吸附法纯化培养OECs,采用荧光染色法鉴定OECs并进行纯度测定;采用ELISA法检测培养上清液中神经生长因子(NGF),脑源性神经营养因子(BDNF)和胶质源性神经营养因子(GDNF)的水平。结果:通过上述方法,可以得到纯度85%以上的OECs,光镜下OECs突起细长,以典型的双极或三极细胞为主,背景有少量扁平的、折光性差的细胞存在;NGF、BDNF和GDNF在上清液中均有存在,其中NGF含量最高,NGF和BDNF含量随着培养时间延长逐渐升高,11 d达到高峰,GDNF仅在培养第11 d后检测到。结论:采用差速贴壁法结合免疫亲和吸附法可以获得较高纯度的OECs,培养11~14 d,OECs数量多且分泌旺盛,适合用于细胞移植。
OBJECTIVE: To create an ideal method to purify olfactory ensheathing cells (OECs) and to observe the content of neurotrophic factor in the supernatant of OECs at different time points, so as to provide the theoretical basis for choosing the optimal timing of OECs transplantation. Methods: OECs were purified by differential adherence method and immunoaffinity adsorption method. OECs were identified by fluorescence staining and purity was determined. Nerve growth factor (NGF), brain-derived neurotrophic Factor (BDNF) and glial derived neurotrophic factor (GDNF) levels. Results: The OECs with the purity above 85% could be obtained by the above method. The OECs were slender and elongated under the light microscope. The bipolar or tripolar cells were dominated by typical bipolar or tripolar cells with few flat and poorly refracted cells in the background. NGF, BDNF And GDNF were present in the supernatant. Among them, the highest content of NGF, the content of NGF and BDNF increased gradually with the prolongation of culture time and reached the peak on the 11th day. GDNF was only detected on the 11th day after culture. Conclusion: OECs with high purity can be obtained by differential adherence method combined with immunoaffinity adsorption. After cultured for 11-14 days, OECs are abundant in number and highly secreted and suitable for cell transplantation.