Proteomic Profiling of Stem Cell Differentiation

描述(申请人提供)：骨髓间充质干细胞(BMMSC)具有分化为不同类型细胞的潜力。本研究的长期目标是在体外控制BMMSC的分化，并利用BMMSC来源的平滑肌细胞(SMC)构建组织工程血管移植物。血管壁上的周期性机械应变和转化生长因子13在SMC分化和血管重塑中起重要作用。研究人员发现，机械应变和转化生长因子-I31均可增加骨髓间充质干细胞中SMC标志物的表达。然而，骨髓间充质干细胞分化过程中的蛋白质组学变化及其机制还不是很清楚。蛋白质组学为研究干细胞功能提供了一种系统而有力的方法。研究人员假设：(1)BMMSC及其分化可以通过表达特异的蛋白质标志物来表征；(2)机械应变和转化生长因子-β]通过不同的机制调节BMMSC的分化，并协同BMMSCs向SMC分化。在这项探索性/发展性研究中，提出了两个具体目标。在目标1中，研究人员将确定BMMSC的蛋白质组学特征，并确定BMMSC的潜在标记。转化生长因子-β]等分化因子将用于诱导骨髓间充质干细胞的蛋白质组改变和分化。使用最先进技术的综合策略将被用于蛋白质组分析。二维凝胶电泳法和多维液相色谱技术将用于蛋白质/多肽的分离。质谱学将用于蛋白质的鉴定和表征。将生成BMMSC的参考地图。只在未分化的骨髓间充质干细胞中表达的蛋白质将被鉴定。在目标2中，研究人员将确定骨髓间充质干细胞在机械应变和转化生长因子-B作用下的蛋白质组变化。表达和翻译后修饰发生变化的蛋白质将被识别。固定化金属亲和捕捉法将被用于磷酸肽的富集化。将确定骨髓间充质干细胞分化过程中的整体机械力化学信号转导，并将为机械应变和转化生长因子-β差异调节、共享和协同的信号通路生成MAP。将进行分类和聚类分析以将蛋白质和信号通路关联起来。本研究将促进我们对骨髓间充质干细胞蛋白质组和分化的认识，并为今后更有针对性和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大影响，为构建用于血管组织修复的骨髓间充质干细胞工程提供合理的依据。

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.