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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。
OBJECTIVE: To compare the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) induced by recombinant human amelogenin (rhAm) and enamel matrix proteins (EMPs) To investigate the regulation mechanism of rhAm on hBMSCs osteogenesis and to provide a theoretical basis for the clinical application of rhAm. Methods: The full length rhAm of 25 kDa was induced and purified by induction. Pig EMPs were purified by acetic acid method and hBMSCs were primary cultured in vitro. Real-time quantitative PCR and Western blotting were used to detect the changes of osteoblasts (Runx2, ALP, COL-I) after rhBMCs were treated with rhAm and EMPs at different time points. The effects of two proteins on the osteogenesis and mineralization of hBMSCs were detected by alkaline phosphatase and alizarin red staining. Data were statistically analyzed using SPSS 13.0 software package. Results: Primary hBMSCs were obtained in vitro. Real-time quantitative PCR, Western blotting and cell staining showed that 10μg / ml rhAm and 200μg / ml EMPs could significantly promote the gene and protein expression of osteogenesis-related factors in hBMSCs, and this effect was related to the protein action time Sex. CONCLUSION: Both rhAm and EMPs can significantly promote the osteogenesis of hBMSCs, with a certain time-dependent effect.