成年大鼠嗅鞘细胞的分离培养及形态学研究

目的：探讨成年大鼠嗅鞘细胞（olfactory ensheathing cells，OECs）的分离培养和纯化方法，并观察其形态学变化及免疫组织学特性，为研究脊髓损伤后神经再生获得丰富嗅鞘细胞来源奠定理论基础。方法：采用原代培养技术，从成年SD大鼠的嗅球分离培养嗅鞘细胞，用Nash差速贴壁法和阿糖胞苷抑制相结合的方法培养纯化，倒置相差显微镜下观察嗅鞘细胞的形态变化特点及其生长情况，并行神经生长因子受体（NGFRp75）和胶质纤维酸性蛋白（GFAP）免疫组织化学染色鉴定以及纯度检测。结果：体外培养的成年大鼠嗅鞘细胞主要为双极或三极细胞，其突起细长。未经纯化则成纤维细胞生长迅速而占据优势，而纯化培养后的嗅鞘细胞纯度可达90％以上。免疫细胞化学染色显示，培养的嗅鞘细胞呈现NGFRp75和GFAP染色阳性。结论：成年大鼠嗅鞘细胞的原代培养中细胞纯化是必要且必须的；而在脊髓损伤修复的研究中，Nash差速贴壁法和化学药物法不失为一种简单、经济、实用的嗅鞘细胞纯化方法。

Primary Culture and Morphologic Study of Olfactory Ensheathing Cells From the Aadult Rat Olfactory Bulb

Objective： To culture and purify the olfactory ensheathing cells （OECs） from the aduh rat olfactory bulb in vitro, and to investigate the morphological changes and the immunohistologic properties of the cultured OECs, to obtain the abundant OECs for axon regeneration after spinal cord injury. Methods： The OECs were dissociated from the adult rat olfactory bulbs. The primarily cultured OECs were purified based on the different rates of attachment of the various harvested cell types and Ara-C inhibition. The morphological changes of the cultured OECs were observed under a phase contrast microscope at different culture periods. The OECs were identified by GFAP and NGFRp75 with immunocytochemistry staining and purity detection. Results： The OECs displayed a very characteristic morphological appearance. Most of OECs were bipolar or tripolar with long and thin processes. In the unpurified group the rapidly proliferating fibroblasts were in the majority after 10 days in culture. The OECs purification was estimated to be 90% after 14 days in culture and the immunocytochemistry results of GFAP and NGFRp75 were both positive. Conclusion： The purification is necessary and imperative in primary culture of OECs from the adult rat olfactory bulbs. The method of purification for OECs based on the differing rates of attachment of the various cell types and Ara-C inhibition is simple, inexpensive and practical in the research of the regeneration after spinal cord injury.