人牙髓干细胞的体外分离、培养及鉴定

Isolation, culture and identification of dental pulp stem cell

目的 :体外分离、培养人牙髓干细胞 ,从不同角度对其进行鉴定。方法 :采用胶原酶和Dispase酶联合消化法培养人牙髓干细胞 ,有限稀释法分离纯化 ,将克隆形成的细胞通过透射电镜观察其超微结构、流式细胞仪细胞周期分析及抗波形丝蛋白 (Vimentin)、CD44、骨粘素 (ON)、牙本质涎蛋白 (DSP)免疫组化染色方法进行鉴定。结果 :通过有限稀释法获得了人牙髓干细胞克隆 ,透射电镜下观察这种克隆形成细胞具有干细胞典型的超微结构特点 ,大多数细胞处于G0 /G1期 ,为细胞周期的静止期 ,并具有牙源性未分化间充质细胞的表型特点。结论 :克隆化培养是人牙髓干细胞较为有效的分离纯化方法 ,该方法分离的人牙髓干细胞具有干细胞的结构、细胞周期及表型特点。

Objective:to isolate and identify the dental pulp stem cell from human third molar.Method: dental pulp tissue was digested with mixture of collagenase and dispase and the stem cells were isolated by limited dilution of culture cell for single cell clone. The transmission electron microscopy(TEM), flow cytometry and immunohistochemistry proceduse were employed to detect the ultrastrcture,cell cycle and surface marker of the cloning cell, respectively.Result: the cloning cell showed the characteristic of undifferented cell at the ultrastructural level; most of them were in quiescence based on ratio of G0/G1 phase and they positively expressed surface protein of Vimentin, CD 44 ,ON and negatively expressed that of DSP .Conclusion: cloning incubation may be the effective way to isolate and purify the dental stem cell and the isolated cells were self-renewal, undifferention, quiescent dental mesenchymal origination, they are dental pulp stem cell.