染氟成骨细胞的双向电泳和质谱鉴定

目的探讨双向电泳和质谱鉴定技术在染氟成骨细胞蛋白表达变化中的意义,为探索氟骨症发病机制提供有效手段。方法采用小鼠乳鼠颅骨来源的成骨细胞进行培养,抽提染氟(2mg/L)72h组和对照组成骨细胞蛋白,用双向凝胶电泳进行分离及ImageMaster2DElite软件分析电泳图谱,基质辅助激光解吸电离-飞行时间质谱仪对两组间比较具有统计学意义的差异蛋白点进行鉴定。结果在对照组成骨细胞蛋白双向电泳图谱上可观察到671个蛋白点,而在染氟组双向电泳图谱上可见到837个蛋白点;在染氟成骨细胞表达有明显差异的蛋白点经质谱鉴定出12种(7种蛋白表达增多,5种降低),主要是与细胞代谢旺盛、蛋白质氧化折叠和氧化应激等相关的蛋白。结论实验所采用的双向电泳和质谱鉴定方法可有效分离和鉴定成骨细胞蛋白点,为氟骨症的发生机制提供了有价值的新线索,表明蛋白质组学技术较其他方法具有很大的优越性。

Analysis of proteins in osteoblast exposed to fluoride by two-dimensional electrophoresis and mass spectrometry

Objective To study the role of osteoblast proteomics exposed to fluoride using two-dimensional electrophoresis(2-DE) and mass spectrometry and to explore the mechanism of skeletal fluorosis. Methods Calvarial osteoblast out of neonatal mouse were cultured and exposed to fluoride(2 mg/L)72 h. The osteoblast proteins were separated using immobilized pH gradients 2-DE. Image analysis was carried out using Image Master 2-DE Elite software. The significantly differential proteins were identified by matrix assisted laser adsorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Results The maps of 2-DE showed that 671 protein spots were visualized in osteoblast of control group and 837 protein spots in fluoride-treated cells. The significantly differential proteins identified by MALDI-TOF MS added up to 12 kinds, mainly associated with vigorously intracellular metabolism, protein oxidative folding and oxidative stress, among which 7 were up-regulated and 5 were down-regulated. Conclusions The methods of 2-DE and MALDI-TOF-MS utilized in our study have effectually separated and identified the osteoblast proteins, and provided a valuable clue to study the mechanism of skeletal fluorosis. It is suggested that proteomical techniques are powerful methodologies for the better understanding of skeletal fluorosis.