体外培养人牙髓干细胞的生物学特性

Biological Characteristics of Cultured Human Dental Pulp Stem Cells in Vitro

目的研究体外培养的人牙髓干细胞的生物学特性。方法收集因正畸拔除的第一前磨牙,用酶消化法分离能够形成克隆且具有高增殖力的细胞,细胞以低密度接种传代培养;免疫荧光法检测第3代细胞表面标志的表达;取第4代细胞,用含有20%胎牛血清、地塞米松、β甘油磷酸钠和维生素C的培养液诱导3周,茜素红染色观察诱导后细胞矿化情况,免疫荧光法检测牙本质涎蛋白(DSP)表达,改良Kap low氏法检测诱导前后碱性磷酸酶的表达。结果诱导前细胞表达Stro-1、Cbfa-1、和Ⅰ型胶原,碱性磷酸酶呈弱表达,不表达DSP;诱导后细胞表达DSP,碱性磷酸酶呈强表达,并可见钙结节形成。结论分离所得细胞具有较高的增殖和分化能力,与间充质干细胞具有相似的表面标志,并在诱导后表达DSP而具备牙髓干细胞的特征。

Objective To study the biological characteristics of cultured human dental pulp stem cells in vitro. Methods Human healthy first premolars extracted for orthodontic reasons were collected. Clonogenic and highly proliferative cells were derived from enzymatically disaggregated adult human dental pulp. Cells were implanted in a six- well plate at a low density. Clonogenic cells were isolated and tested for surface markers. They were also induced by Dulbeco＇s modified essential media containing 20% FBS, dexamethasone, β sodium glycerophosphate and Vitamine C for 3 weeks. The induced and uninduced cells were tested by immunohistochemistry. Results The uninduced cells isolated from human dental pulp positively expressed protein of stro-1, cbfa-1, and collagen I, but did not express dentin sialoprotein （DSP）. Alkaline phosphatase was strongly expressed after 3 weeks induction. The induced cells also expressed relatively specific odontoblasts marker DSP. Calcium deposite was formed after 3 weeks induction. Conclusion The cells isolated from human dental pulp have a high ability to proliferate and differentiate. They express some surface markers similar to mesenchymal stem cells and DSP can be expressed after induction.