嗅感觉神经细胞的分离与培养

The primary culture of olfactory receptor neurons

目的 建立哺乳动物嗅感觉神经细胞体外培养的方法。方法取成年家兔的嗅区粘膜,用胰酶和胶原酶消化法进行嗅感觉神经细胞的分离与原代培养,并应用抗神经微丝蛋白(NFP)抗体、抗嗅觉标记蛋白(OMP)抗体进行免疫细胞化学染色,同时采用甲苯胺蓝神经特异性染色和扫描电镜对其进行鉴定。结果 从嗅粘膜分离以及原代培养得到的嗅感觉神经元,光镜、电镜下为典型的双极神经元,OMP、NFP阳性,神经元特异性染色阳性。分离的嗅感觉神经元可在体外存活数小时,其细胞形态、生存数量和存活时间均能满足体外实验的基本要求。结论 本实验在家兔建立了较稳定、可靠的嗅感觉神经细胞分离和原代培养方法。为深入开展嗅感觉神经细胞的离体研究,提供了较为理想的材料来源和技术保障。

Objective To develop a protocol of ol-factory receptor neurons (ORNs) primary culture in adult mammals. Methods The isolation and primary culture of ORNs were got by trypsin and collagenase from olfactory mucosa of adult rabbits. The process of cellular growth and differentiation of ORNs were observed by in-verted phase contrast microscopy and scanning electron microscopy.Immunocytochemical identification of ul-tured ORNs was performed by monoclonal antibody of neurofilament protein and polyclonal antibody of olfacto-ry marker protein. Nerve special toluidine blue staining was also performed.Results The dissociated ORNs of adult rabbits could survive well and differentiate into ma-ture phenotype under the serum in vitro. The celi amount, celi morphological shape and surviving period of ORNs were able to meet the requirements of cvtological experi-ments.Conclusion We have developed a constant and reliable method in isolation and primary culture ORNs of the adult rabbits.The method extend the material sources of ORNs and provides the technical guarantee for further study in ORNs in vitro.