成牙骨质细胞体外矿化培养相关蛋白的时序性表达

Sequential Expression of Mineralization Related Proteins in Cementoblast During Mineralized Culture in Vitro

目的 检测骨涎蛋白 (BSP)、硷性磷酸酶 (ALP)、及牙骨质特异性蛋白—牙骨质附着蛋白 (CAP)在成牙骨质细胞 (CB)体外矿化培养过程中的时序性表达 ,并研究这一过程中CB的形态和生物学特征。方法 将CB接种在盖玻片上 ,分别培养 6、12h、1～ 6d后 ,取出盖玻片 ,应用免疫细胞化学的方法检测这三种蛋白在CB上的表达。结果 CB接种在盖玻片 6h后 ,在CB胞浆内三种蛋白都有表达 ;在 2、3d ,细胞聚集形成分层状 ,BSP、ALP表达减弱 ,CAP不表达 ;4d后 ,聚集的细胞增殖并分化 ,形成具有矿化功能的细胞结节。此时 ,3种蛋白的表达强弱不等 ,BSP、ALP在结节细胞表达强 ,在周围细胞表达弱 ,而CAP只在结节细胞表达 ,在周围细胞不表达。之后的 10～ 16d ,细胞结节进一步形成矿化结节。结论 本实验揭示了CB在体外增殖、分化及矿化过程中 ,CAP ,BSP和ALP的表达情况及细胞形态的变化。为CB在体内的增殖、分化、矿化及CB在体内形成牙骨质的研究提供了一些参考。

Objective To investigate sequential expression of bone sialoprotein(BSP), alkaline phosphatase(ALP) and cementum attachment protein(CAP) in cementoblast(CB) during mineralized culture in vitro, and study the morphological and biologic characters of the CB in this process. Methods CB was seeded on the glass coverslips, and cultured for 6h, 12h, 1d, 2d, 3d, 4d, 5d, 6d, 8d, 10d, 12d and 16d, respectively. The expression of BSP, ALP and CAP proteins was detected using immunocytochemical method. Results 6 hours after plated, cementoblast expressed all of the three proteins. In the second and third days after plated, the cells became confluent and formed multiple layers, BSP and ALP expression decreased, and CAP did not be expressed at all. From the forth day, the cells formed cell nodules with mineralizing function. The cells in the nodules strongly expressed BSP,ALP and CAP, the cells around the nodule weakly expressed BSP and ALP, and did not express ALP. During the following 10 to 16 days, the cell nodules became mineralized nodules. Conclusion These results elucidated the changes of ALP, BSP and CAP expression as well as cell morphology during the CB proliferation, differentiation and mineralization in vitro, and provided some valuable information for studying the formation of cementum and CB proliferation, differentiation and mineralization in vivo.