【摘要】 目的:嗅鞘细胞纯化培养方法有免疫亲和吸附法、免疫磁珠法、化学药物法、差速贴壁法、无血清饥饿法等,但均存在缺陷。比较3种纯化方法原代培养人胚嗅球嗅鞘细胞的纯度,寻求简单实用的高纯度人胚嗅球嗅鞘细胞的体外培养方法。方法:实验于2007-08/12在西安交通大学口腔医院医学实验中心进行。选用流产胚胎标本24份,胎龄4~6月,由西安交通大学第二医院和陕西省妇幼保健院产科提供。实验经产妇及家属同意,并经医院伦理委员会的批准。实验方法:取胚胎嗅球分离培养嗅鞘细胞,制成单细胞悬液。将单细胞悬液加入未包被的培养瓶中培养,然后再次将细胞悬液加入未包被的培养瓶中,具体差速时间根据观察决定。最后将差速贴壁后含未贴壁细胞的细胞悬液加入多聚赖氨酸包被的培养瓶或培养板中,用3种方法纯化培养:阿糖胞苷法、无血清饥饿法和间断神经营养因子3法纯化培养人胚嗅球嗅鞘细胞。比较3种纯化方法下嗅鞘细胞生长变化及纯度。结果:①培养早期嗅鞘细胞呈圆形,随着培养过程进行细胞逐渐伸出细长突起呈双极、三极和多极细胞,胞核位于细胞中央。成熟的嗅鞘细胞大多呈双极、三极细胞与许旺细胞相似,细胞折光性好,胞核呈圆形位于细胞中央,"煎蛋样"细胞少见。②差速贴壁法培养3~6d时纯度为88%,以后成纤维细胞增殖嗅鞘细胞纯度迅速下降。阿糖胞苷法阿糖胞苷作用后细胞大量死亡,免疫染色几乎未见阳性反应细胞存在。无血清饥饿法培养3~6d时嗅鞘细胞纯度为90%,但细胞状态差。间断神经营养因子3法培养3~9d时嗅鞘细胞纯度为95%,且细胞状态良好。结论:差速贴壁结合间断间断神经营养因子3法比结合阿糖胞苷法、无血清饥饿法获得的嗅鞘细胞纯度高,且细胞状态良好。更多还原

【Abstract】 AIM: Olfactory ensheathing cells (OECs) culture method includes immune affinity adsorption, imunomagnetic beads method, chemicals method, differential adherence method, starvation method without serum and so on. These methods possess shortcomings. This study searched a high purity of human fetal olfactory bulb OECs in vitro culture method by comparing three different methods. METHODS: The experiments were undergone at the Experiment Center of Stomatology Hospital of Xi’an Jiaotong University from August to December 2007. Totally 24 induced abortion embryo (conceptus age is 4-6 months) were offered by Second Affiliated Hospital of Xi’an Jiaotong University and Department of Obstetrics of Shanxi Maternal and Child Health hospital. The experiments were admited by the puerperant and her family and were licensed by Ethics committee of the Hospital. Olfactory bulb was abstracted to culture the human fetal OECs, and the monoplast suspension were made and cultured in the uncoated culture flask. Cell suspension was added in the uncoated culture flask again. Differential time depended on observation. After attachment, cell suspension containing unattached cells was removed to culture bottle or culture plate coated with polylysine. Cytarabine method, starvation method without serum and NT3 method without serum were used to observe human fetal OECs. Growth and purity of OECs were compared by three different culture methods. RESULTS: ①OECs in early culture phase were round, and then showed dipolar, tripolar shape and multipolar cells. Nucelus was in the middle of the cell. Mature OECs were Schwann’s cells like with good refraction. Nuclei were round in the middle of cells, and no "fried egg" shape cells appeared. ②The purity of OECs could reach 88% with differential adhesion method from 3-6 days, and then fibroblasts would proliferate quickly and reduce the purity of OECs. After cytarabine method, a large number of cells died and no positive reaction cells were found by immunostaining. By serum-free starvation method, the OECs could reach a higher purity 90%, but cells condition was bad from 3-6 days. By the NT3 methods without serum for 3-9 days, the purity could touch the highest score 95% with a good cell condition. CONCLUSION: By NT3 without serum methods will give a higher purity of OECs in a long time than the other 2 methods and have a good cell condition.更多还原