骨髓间充质干细胞定向诱导成骨分化的蛋白质组学分析

Proteomic analysis of human mesenchymal stem cells undergoing osteoblast differentiation

目的通过对人骨髓间充质干细胞(hBMSCs)定向诱导成骨细胞分化过程中不同分化阶段的比较蛋白质组学分析,寻找与干细胞分化的启动、进展及成熟有关的功能蛋白质,为研究干细胞的调控分化提供线索。方法采用贴壁培养法从人骨髓中分离获得hBMSCs,体外扩大培养,以成骨诱导培养体系诱导BMSCs定向成骨细胞分化,选取诱导的1、7、14、21d,分别提取未诱导细胞及诱导组细胞的总蛋白,采用荧光差异凝胶电泳技术分离各组总蛋白,图像分析检测差异表达的蛋白质点,经酶切后行蛋白肽质量指纹谱和数据库检索,鉴定差异蛋白表达。结果贴壁细胞于3d左右开始伸出突起,2～6d时细胞增殖较缓慢,逐渐长为梭形,7d开始细胞增殖迅速,细胞呈漩涡状生长,15d左右逐渐达到完全融合。碱性磷酸酶染色鉴定BMSCs向成骨诱导分化,BMSCs染色阴性,成骨细胞阳性。BMSCs与成骨细胞凝胶图像存在多个蛋白差异点,选取45个差异点,切胶作质谱分析。经质谱分析和数据库检索后鉴定到23个差异表达蛋白质点,大多数差异点蛋白的功能涉及细胞骨架形成、能量代谢、信号转导等过程。结论应用蛋白质组学方法,从功能蛋白质组学的层面阐述BMSCs定向诱导成骨分化相关的重要蛋白的变化,可以为更深入揭示BMSCs定向分化的分子机制提供依据。

Objective To find the functional proteins related to the start, progress and maturation of stem cell differentiation through the proteomic analysis of the human bone marrow-derived mesynchymal stem cells （hBMSCs） undergoing osteoblast differentiation, and to provide clues for the regulation and control of stem cell differentiation. Methods BMSCs were isolated and expanded in vitro through adhesion culture. Total proteins of the undifferentiated cells and osteogenic differentiated cells over a period of 21 days were extracted and subjected to proteomic analysis including fluorescent differential gel electrophoresis, peptide mass fingerprinting and database searching. Results A total of 23 differentially expressed protein spots were identified by mass spectrometry analysis and database searching, most of which were involved in cytoskeleton, energy metabolism and signal transduction processes, Conclusions Functional proteomic analysis could provide theoretical basis for revealing the molecular mechanism of BMSCs differentiation.