比格犬牙髓干细胞的培养与鉴定

Isolation and identification of Beagle dog dental pulp stem cells

目的应用经典的人牙髓干细胞分离培养方法证明比格（Beagle）犬牙髓干细胞的存在，以期为大型动物的牙源性干细胞性质及其应用研究提供基础。方法获取比格犬健康恒牙牙髓组织，应用分散酶进行消化培养获得细胞，并通过观察其生物学特性、多向分化能力及干细胞相关表面标志物的表达等方法进行鉴定。结果经酶消化法获得的比格犬牙髓干细胞形态与人牙髓干细胞形态相似，呈典型的成纤维细胞形态；比格犬牙髓干细胞的牙龈成纤维细胞集落形成率为150个集落／10。个细胞，显著高于人牙髓干细胞的牙龈成纤维细胞集落形成率（60个集落／10。个细胞），差异有统计学意义（P〈0．001）；比格犬牙髓干细胞具有多向分化能力，在体外经诱导可以形成矿化结节、脂滴和成软骨细胞，同时比格犬牙髓干细胞表达间充质干细胞相关表面标志物基质细胞抗原（STRO-1）、CDl46、碱性磷酸酶、波形蛋白、细胞角蛋白18及神经上皮干细胞蛋白（nestin）。结论本项研究证实了比格犬牙髓组织中存在牙髓干细胞，并具有增殖活跃的特性和多向分化的潜能。

Objective To identify the existence of the dental pulp stem cells in Beagle＇s pulp tissue by using the same methods of isolating and culturing the human dental pulp stem cells. Methods Pulp tissue was extirpated from the crown and root of the Beagle＇s healthy permanent tooth, and digested by dispase for cell culture. Classical identification methods of mesenchymal stem cells including observation of biological characteristics, capacity of multilineage differentiation, and expression of specific markers associated with mesenchymal stem cells were applied to verify the existence of Beagle＇s dental pulp stem cells. Results A clonogenic, rapidly proliferative population of ceils were isolated from Beagle＇ pulp tissue. Under the same culture condition, the Beagle＇s dental pulp stem ceils had a significant higher colony- forming unit-fibroblast （CFU-F） formation rate （ 150 colony/104 cells ） than the dental pulp cells derived from the human pulp tissue （60 colony/lO4 cells ）. These cells also had the multilineage differentiation ability. They could be induced to form mineralized nodules, lipid droplets and chondrocytes. Furthermore these cells expressed the mesenchymal stem cell markers including STRO-I, CD146, alkaline phosphatase, nestin, vimentin and cytokeratin-18. Conclusions There are dental pulp stem cells in the Beagle＇s pulp tissue.