Rheumatology Division, ProteoRed/ISCIII Proteomics Group, INIBIC-Hospital Universitario de A Coruña, 15006-A Coruña, Spain.
J Proteome Res. 2012 Nov 2;11(11):5350-61. doi: 10.1021/pr300572r. Epub 2012 Oct 3.
Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.
人骨髓间充质干细胞（hMSCs），不仅存在于骨髓中，也存在于关节滑膜衬里中，可被诱导向软骨细胞分化。因此，hMSCs 在修复骨关节炎（OA）和其他关节疾病的软骨缺损方面具有巨大的治疗潜力。然而，软骨生成过程的分子机制在一定程度上仍然未知。在这项工作中，我们首次应用稳定同位素标记的氨基酸在细胞培养中的技术（SILAC）来定量分析 hMSCs 软骨分化过程中的蛋白质调节。首先，我们对骨髓分离的间充质干细胞（hBMSCs）进行了代谢标记程序的标准化，并评估了这些条件下发生的软骨生成质量。然后，在这些标记的细胞上诱导软骨分化，并采用定量蛋白质组学方法评估两个分化日之间的蛋白质变化。通过这种策略，我们可以通过 LC-MALDI-TOF/TOF 分析鉴定 622 种不同的蛋白质，并发现 65 种蛋白质的丰度在软骨生成的第 2 天和第 14 天之间有显著变化。进行免疫组织化学分析以验证在一组六个在细胞中发挥不同生物学作用的蛋白质上的变化：纤连蛋白、凝胶蛋白、波形蛋白、α-ATP 酶、线粒体超氧化物歧化酶和亲环蛋白 A。与软骨诱导的第 2 天相比，所有这些蛋白质在第 14 天都增加了，因此是增强细胞外基质合成、细胞黏附、代谢和对软骨生成早期发生的应激反应过程的标志物。我们的策略使我们对特定蛋白质功能和软骨生成机制有了更深入的了解，并提供了 hBMSCs 中这种分化过程的蛋白质标志物。
