Hannover Medical School, Institute of Toxicology, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Rapid Commun Mass Spectrom. 2010 Mar 15;24(5):561-70. doi: 10.1002/rcm.4412.
Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration.
多能造血祖细胞（HPC）系 DKmix 的给药改善了心肌梗死后的心脏功能，并通过旁分泌机制加速了皮肤伤口愈合。本研究的目的是分析 DKmix 细胞分泌的蛋白质，以鉴定负责旁分泌的因子，并评估其与广泛治疗效果的相关性。使用基于质谱（MS）的方法来鉴定 DKmix 细胞分泌的蛋白质。收集 DKmix 条件培养基的无血清培养上清液，并分离其中存在的蛋白质，用胰蛋白酶消化，然后通过基质辅助激光解吸/电离串联飞行时间（MALDI-TOF/TOF）MS 分析得到的肽。总共鉴定出 95 种不同的蛋白质。其中，鉴定出分泌蛋白半乳糖凝集素-3 和凝胶蛋白。这些蛋白质已知可刺激细胞迁移并影响伤口愈合和心脏重塑。其余的蛋白质来源于细胞质（69%）、核（12%）、线粒体（4%）和细胞质膜（3%）等细胞内区室，表明条件培养基中的 DKmix 细胞是可渗透或漏出的。此外，还使用夹心免疫测定法来检测和定量细胞因子和趋化因子。检测到白细胞介素-6（IL-6）、白细胞介素-13（IL-13）、单核细胞趋化蛋白-1（MCP-1）、单核细胞趋化蛋白-3（MCP-3）、单核细胞趋化蛋白-1alpha（MIP-1alpha）和单核细胞趋化蛋白-1beta（MIP-1beta），浓度较低。本研究鉴定出一组存在于 DKmix 条件培养基中的蛋白质，它们作为组织修复的旁分泌调节剂发挥作用。此外，这表明 DKmix 衍生的条件培养基可能通过促进组织再生而具有治疗潜力。
