间充质干细胞定向成骨细胞分化的蛋白质组学分析.docVIP

间充质干细胞定向成骨细胞分化的蛋白质组学分析 【关键词】 蛋白质组学；间质干细胞；蛋白表达 　　Proteomic analysis of human bone marrowderived mesenchymal stem cells induced to differentiate into osteoblasts 　　【Abstract】 AIM: To analyze the difference of protein profiles between human bone marrowderived mesenchymal stem cells (MSCs) and osteogenic differentiated MSCs by proteomic approach. METHODS: The MSCs were isolated primarily from bone marrow of adults and detected in cell purity by flow cytometry analyzing cell phenotypes and in cell function by multilineage potential test. The undifferentiated MSCs and osteogenic differentiated MSCs over a period of 14 d were collected and cell total proteins were extracted for finding out the differential expression by proteomic analysis including 2D gel electrophoresis and Peptide Mass Fingerprinting technology. RESULTS: Twentythree differentially expressed proteins were identified by MALDITOFMS analysis. CONCLUSION: Proteomic approach is a high throughput method to screen the key proteins which play important roles in MSCs osteogenic differentiation. 　　【Keywords】 proteomics; mesenchymal stem cells; protein expression 　　【摘要】 目的: 探讨用蛋白质组学方法分析人骨髓间充质干细胞（MSCs）定向成骨细胞诱导分化中细胞蛋白表达谱的改变. 方法: 从人骨髓细胞中分离获得MSCs，经细胞表型及多向分化能力测定鉴定MSCs的纯度和功能后，采用定向成骨细胞分化诱导培养体系，于分化后14 d分别提取未分化细胞及分化后细胞的总蛋白，双向电泳分析，确定蛋白差异点，经酶切后行蛋白肽质量指纹谱鉴定差异蛋白表达. 结果: 双向电泳找到38个蛋白差异点，质谱分析鉴定出23个差异蛋白分子表达. 结论: 用蛋白质组学方法可高通量筛选与MSCs定向诱导成骨细胞分化相关的重要蛋白. 　　【关键词】 蛋白质组学；间质干细胞；蛋白表达 　　0引言 　　间充质干细胞(mesenchymal stem cells, MSCs)是一群具有向成骨细胞、成软骨细胞和脂肪细胞分化能力的干细胞，存在于成体的骨髓、骨、脂肪和肌肉等组织中. MSCs体外贴壁性使之易于分离纯化，且体外培养的MSCs增殖能力强，是一种具有重要应用价值的种子细胞［1］. 但有关MSCs定向分化的分子机制尚不清楚. 为探讨MSCs定向成骨分化过程中细胞内可能具有调控作用的蛋白分子的变化，我们首先从人骨髓单个核细胞中分离获得MSCs，对所获得的MSCs进行了细胞表型及多向分化能力测定，在鉴定MSCs的纯度和功能后，采用定向成骨分化诱导培养体系诱导MSCs定向成骨细胞分化，对未分化MSCs及分化后细胞总蛋白进行提取，经双向电泳比较蛋白差异点，选择差异点蛋白，酶切后进行蛋白点肽质量指纹谱鉴定分析. 　　1材料和方法 　　1.1材料 　　骨髓取自食道癌手术患者，年龄56～67岁. 主要试剂Dulbeccos modified Eagles medium（DMEM）培养基为Gibco产品，胎牛血清为Stem cell产品，Percoll为Amersham Pharmacia产品，CD34，CD45，CD73，CD10