单次差速贴壁法纯化培养大鼠嗅神经鞘细胞的实验研究

Purification and cultivation of olfactory ensheathing cells from olfactory bulbs in rats by single adhering to culture plastic in different time technique

目的:探讨单次差速贴壁法纯化培养成年大鼠嗅球嗅鞘细胞的可行性。方法:选取8～10周龄的成年SD大鼠,无菌条件下切取嗅球、剪碎,胰酶消化制成细胞悬液后接种,采用18h单次差速贴壁的方法纯化培养嗅鞘细胞,定期在倒置显微镜下观察嗅鞘细胞的形态学变化及生长情况,并对其行神经生长因子受体p75抗体(NGFRp75)及碘化丙啶的免疫荧光鉴定,同时计算嗅鞘细胞的纯度。结果:通过18h单次差速贴壁法纯化培养的嗅鞘细胞经NGFRp75免疫荧光鉴定后,纯度可达70%～80%,形态上以双极和三极细胞为主,伴有少许单极及多极细胞。结论:应用18h单次差速贴壁法纯化培养嗅鞘细胞是一种简便、稳定、经济、快捷的方法。

Objective To investigate the feasibility of purification and cultivation of olfactory ensheathing cells （OECs） from olfactory bulbs in rats by single adhering to culture plastic in different time technique. Methods The olfactory bulbs were harvested from adult SD rats （8 - 10 weeks old）, cut into pieces, digested by pancreatin, then the cells were cultured in DMEM/F12 medium with 10% fetal calf serum, and purified and cultured by single adhering to culture plastic in different time technique. The purified OECs were observed by inverted microscope regularly, and the neural growth factor receptor p75 （NGFR p75） and propidium iodide were identified by immunofluorescence staining. The purity of olfactory ensheathing cells was calculated. Result The purity of OECs was about 70% to 80%. There were many bipolar and tripolar cells, and a few multipolar cells which were all confirmed as the OECs.Conclusion Single adhering to culture plastic in different time technique is a good, simple, and effective way to culture and purify olfactory ensheathing cells.