Institute of Tissue Transplantation and Immunology, Key Laboratory of Ministry of Education for Genetic Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Mol Cell Proteomics. 2010 Mar;9(3):550-64. doi: 10.1074/mcp.M900243-MCP200. Epub 2009 Dec 15.
The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.
多能间充质干细胞（MSCs）的软骨生成潜力使其成为软骨缺陷细胞治疗的有前途的来源；然而，MSCs 软骨生成和自我更新的确切细胞内分子机制在很大程度上仍然未知。为了更深入地了解潜在的分子机制，我们应用等重同位素标签相对和绝对定量（iTRAQ）标记与在线二维 LC/MS/MS 技术相结合，鉴定体外软骨生成模型中差异表达的蛋白质：C3H10T1/2 细胞（一种鼠胚胎间充质细胞系）的软骨分化通过微团培养和 100ng/ml 骨形态发生蛋白 2 处理诱导 6 天。共鉴定出 1756 种蛋白质，平均假发现率<0.21%。定量数据的线性回归分析给出了很强的相关系数：两次二维 LC/MS/MS 分析的相关系数分别为 0.948 和 0.923，三次独立 iTRAQ 实验的相关系数分别为 0.881、0.869 和 0.927（p<0.0001）。在 1753 种定量蛋白质中，有 100 种蛋白质发生了显著变化（95%置信区间），其中 6 种蛋白质进一步通过 Western blot 验证。功能分类表明，上调的 17 种蛋白质主要包含成熟软骨细胞的特征以及参与软骨细胞外基质合成的酶，而下调的 83 种蛋白质主要参与能量代谢、染色质组织、转录、mRNA 加工、信号转导和细胞骨架；除了一些有大量文献记载的蛋白质外，这些发生变化的蛋白质中的大多数都是软骨生成的新蛋白质。最后，在软骨分化的背景下验证了本研究中鉴定的两个新的软骨生成相关蛋白 BTF3l4 和 fibulin-5 的生物学作用。这些数据将为我们更好地理解调节这些复杂生物过程的潜在机制提供有价值的线索，并有助于将 MSCs 应用于软骨再生的细胞治疗。
