人胚嗅粘膜嗅鞘细胞的分离、培养和纯化

Isolation,culture and purification of olfactory ensheathing cells from human fetal olfactory mucosa

目的采用经我们改良的差速贴壁法结合神经营养因子3(NT3)和低浓度血清的培养方法对人胚嗅粘膜嗅鞘细胞进行分离和原代纯化培养,探讨建立嗅粘膜嗅鞘细胞体外培养的方法。方法对差速贴壁后的人胚嗅粘膜嗅鞘细胞交替应用含10%胎牛血清和含2.5%胎牛血清、NT3的DMEM-F12培养基进行原代培养。观察嗅粘膜嗅鞘细胞的形态学变化,采用p75NTR和GFAP免疫细胞化学染色进行鉴定和纯度检测。结果人胚嗅粘膜嗅鞘细胞形态多呈双极、三极,伴有细长的突起。p75NTR和GFAP染色均呈阳性反应,体外培养9d时纯度可达83%。结论差速贴壁法结合低浓度血清和NT3应用可以分离培养出人胚嗅粘膜嗅鞘细胞。

Objective To explore the method for obtaining olfactory ensheathing cells from human fetal olfactory mucosa by cell culture for selective adhesion in the presence of neurotrophin-3 （NT3） and low-concentration serum. Methods The olfactory ensheathing cells were cultured alternatively in DMEM/F12 culture medium containing 10% fetal bovine serum （FBS） and the medium containing NT3 and 2.5% FBS every 72 h. The cells were observed for morphological changes and identified using immunocytochemistry with P75^NIR and GFAP, and the cell purity was estimated. Results The olfactory ensheathing cells from human fetal olfactory mucosa were positive for P75^NIR and GFAP, and in in vitro culture, the cells exhibited dipolar or tripolar appearance with long thin neurites. On the 9th day of cell culture, the purity of the olfactory ensheathing cells reached about 83%. Conclusion The olfactory ensheathing cells can be obtained by in vitro culture for selective adhesion in the presence of NT3 and low-concentration serum.