Proteomic Profiling of Stem Cell Differentiation

描述（由申请人提供）：骨髓间充质干细胞（BMMSC）具有分化为不同细胞类型的潜力。本研究的长期目标是在体外控制骨髓间充质干细胞的分化，并利用骨髓间充质干细胞衍生的平滑肌细胞（SMC）构建组织工程血管移植物。血管壁的循环机械应变和转化生长因子13（TGF-B）在SMC分化和血管重构中起重要作用。研究者已经表明，机械应变和TGF-I31都增加了BMMSC中SMC标志物的表达。然而，BMMSCs在分化过程中的潜在机制和蛋白质组学变化尚不清楚。蛋白质组学分析为研究干细胞功能提供了一种系统而有力的方法。研究者推测：(1)BMMSC及其分化可以通过特异性蛋白标志物的表达来表征；(2)机械应变和TGF-B]通过不同的机制调节BMMSC的分化，协同BMMSC向SMCs的分化。在这项探索性/发展性研究中提出了两个具体目标。在Aim 1中，研究者将确定骨髓间充质干细胞的蛋白质组学特征，并确定骨髓间充质干细胞的潜在标记物。TGF-B]等分化因子可诱导骨髓间充质干细胞的蛋白质组变化和分化。采用最先进技术的综合策略将用于蛋白质组分析。二维凝胶电泳和多维液相色谱将用于蛋白质/肽的分离。质谱法将用于蛋白质鉴定和表征。将生成BMMSC的参考地图。仅在未分化的骨髓间充质干细胞中表达的蛋白将被鉴定。在Aim 2中，研究者将确定BMMSC中响应机械应变和TGF-B的蛋白质组变化。将鉴定表达变化和翻译后修饰的蛋白质。固定化金属亲和捕获将用于磷酸肽富集。将确定BMMSC分化过程中的全局机械化学信号转导，并生成由机械应变和TGF-B差异调节、共享和协同的信号通路图。将进行分类和聚类分析，以关联蛋白质和信号通路。本研究将进一步提高我们对骨髓间充质干细胞蛋白质组学和分化的认识，并为今后更集中、更深入的生物学研究奠定基础。这些目标的成功实现将对干细胞工程产生重要的影响，并为血管组织修复的工程化BMMSC提供合理的基础。

DESCRIPTION (provided by applicant): Bone marrow mesenchymal stem cell (BMMSC) has potential to differentiate into different cell types. The long-term goal of this research is to control BMMSC differentiation in vitro and use BMMSC-derived smooth muscle cell (SMC) to construct tissue-engineered vascular grafts. The cyclic mechanical strain in the vessel wall and transforming growth factor 13(TGF-B) play important roles in SMC differentiation and vascular remodeling. The investigator has shown that both mechanical strain and TGF-I31 increase the expression of SMC markers in BMMSC. However, the underlying mechanisms and the proteomic changes of BMMSCs during differentiation are not well understood. Proteomic profiling provides a systematic and powerful approach to studying stem cell functions. The investigator hypothesizes: (1) BMMSC and its differentiation can be characterized by the expression of specific protein markers, and (2) mechanical strain and TGF-B] regulate BMMSC differentiation through distinct mechanisms and synergize the differentiation of BMMSCs into SMCs. Two specific aims are proposed in this exploratory/development study. In Aim 1, the investigator will determine the proteomic profile of BMMSC and identify potential markers of BMMSC. TGF-B] and other differentiation factors will be used to induce the proteome changes and differentiation of BMMSC. A comprehensive strategy using state-of-the-art technologies will be employed for proteome analysis. 2D gel electrophoresis and multidimensional liquid chromatography will be used for protein/peptide separation. Mass spectrometry will be used for protein identification and characterization. A reference map of BMMSC will be generated. The proteins only expressed in undifferentiated BMMSC will be identified. In Aim 2, the investigator will determine the proteome changes in BMMSC in response to mechanical strain and TGF-B. The proteins with changes in expression and post-translational modifications will be identified. Immobilized metal affinity capture will be employed for phosphopeptide enrichment. The global mechano-chemical signal transduction during BMMSC differentiation will be determined, and a map will be generated for the signaling pathways differentially regulated, shared, and synergized by mechanical strain and TGF-B]. Categorization and cluster analysis will be performed to correlate the proteins and signaling pathways. This study will advance our knowledge on BMMSC proteome and differentiation, and lead to more focused and in-depth biological studies in the future. The successful accomplishment of the goals will have high impact on stem cell engineering, and provide a rational basis for engineering BMMSC for vascular tissue repair.