摘要
目的 :比较全长重组人釉原蛋白(recombinant human amelogenin,rhAm)和猪釉基质蛋白(enamel matrix proteins,EMPs)体外诱导人骨髓基质细胞(human bone marrow stromal cells,hBMSCs)成骨分化的作用,探讨rhAm促进hBMSCs成骨的调控机制,为rhAm临床应用提供理论依据。方法:经诱导表达并纯化得到25 k Da全长rhAm;利用乙酸法提纯猪EMPs,体外原代培养hBMSCs。采用实时定量PCR及Western印迹法检测不同时间点rhAm和EMPs作用hBMSCs后成骨因子(Runx2、ALP、COL-I)的变化,观察时效关系。采用碱性磷酸酶和茜素红染色检测2种蛋白对hBMSCs成骨矿化作用的影响。采用SPSS 13.0软件包对数据进行统计学分析。结果:体外培养获得原代hBMSCs。实时定量PCR、Western印迹及细胞染色结果表明,10μg/m L rhAm和200μg/m L EMPs均可明显促进hBMSCs中成骨相关因子的基因及蛋白表达,且这种效果和蛋白作用时间有一定相关性。结论 :rhAm与EMPs均能明显促进hBMSCs成骨,作用效果具有一定的时间依赖性。
PURPOSE： To compare the effect of recombinant full-length human amelogenin（rhAm） and enamel matrix proteins（EMPs） on differentiation of human bone marrow stromal cells（hBMSCs） into osteoblasts. Meanwhile, to investigate the possible mechanism of rhAm promoting osteogenic differentiation of hBMSCs. METHODS： The hBMSCs were cultured in vitro. The cells were treated with 10 μg/m L rhAm and 200 μg/m L EMPs. The gene and protein expression of Runx2, ALP, Col-I were observed by using RT-PCR and Western blot at different time points. The influence of rhAm and EMPs on mineralization and osteogenesis of hBMSCs were observed by using alkaline phosphatase and alizarin red staining methods. The data was analyzed with SPSS 13.0 software package. RESULTS： Both rhAm and EMPs significantly promoted gene and protein expression of Runx2, ALP and Col-I in hBMSCs. Meanwhile, rhAm and EMPs also facilitated osteogenesis and mineralization of hBMSCs. The effects of two proteins on hBMSCs had no significant difference. CONCLUSIONS： Both 10 μg/m L rhAm and 200 μg/m L EMPs can significantly promote differentiation of hBMSCs into osteoblasts. The rhAm may be used in inducing periodontal tissue regeneration in the future.
出处
《上海口腔医学》
CAS
CSCD
2015年第4期390-394,共5页
Shanghai Journal of Stomatology
基金
国家自然科学基金(81070838
81271156)
上海交通大学医工交叉研究基金(YG2011MS31)~~
关键词
牙周组织再生
釉原蛋白
釉基质蛋白
骨髓基质细胞
Periodontal regeneration
Amelogenin
Enamel matrix proteins
Bone marrow stromal cells
