Orthopaedic Surgery Department, University Hospital of North Norway, Tromsø, Norway.
Cell Transplant. 2011;20(9):1381-93. doi: 10.3727/096368910X550215. Epub 2010 Dec 22.
This study was undertaken to compare the phenotype of human articular chondrocytes (ACs) and bone marrow-derived mesenchymal stem cells (MSCs) after cell expansion by studying the spectrum of proteins secreted by cells into the culture medium. ACs and MSCs were expanded in monolayer cultures for some weeks, as done in standard cell transplantation procedures. Initially, the expression of cartilage signature genes was compared by real-time PCR. Metabolic labeling of proteins (SILAC) in combination with mass spectrometry (LC/MS-MS) was applied to investigate differences in released proteins. In addition, multiplex assays were carried out to quantify the amounts of several matrix metalloproteases (MMPs) and their natural inhibitors (TIMPs). Expanded chondrocytes showed a slightly higher expression of cartilage-specific genes than MSCs, whereas the overall spectra of released proteins were very similar for the two cell types. In qualitative terms MSCs seemed to secrete similar number of extracellular matrix proteins (43% vs. 45% of total proteins found) and catabolic agents (9% vs. 10%), and higher number of anabolic agents (12 % vs. 7%) compared to ACs. Some matrix-regulatory agents such as serpins, BMP-1, and galectins were detected only in MSC supernatants. Quantitative analyses of MMPs and TIMPs revealed significantly higher levels of MMP-1, MMP-2, MMP-3, and MMP-7 in the medium of ACs. Our data show that after the expansion phase, both ACs and MSCs express a dedifferentiated phenotype, resembling each other. ACs hold a phenotype closer to native cartilage at the gene expression level, whereas MSCs show a more anabolic profile by looking at the released proteins pattern. Our data together with the inherent capability of MSCs to maintain their differentiation potential for longer cultivation periods would favor the use of these cells for cartilage reconstruction.
本研究通过研究细胞分泌到培养基中的蛋白质谱，比较了在细胞扩增后人类关节软骨细胞（ACs）和骨髓间充质干细胞（MSCs）的表型。ACs 和 MSCs 在单层培养物中扩增数周，如标准细胞移植程序中所进行的那样。最初，通过实时 PCR 比较软骨特征基因的表达。应用代谢标记蛋白质（SILAC）与质谱（LC/MS-MS）相结合的方法来研究释放蛋白质的差异。此外，还进行了多重分析以定量几种基质金属蛋白酶（MMPs）及其天然抑制剂（TIMPs）的含量。与 MSCs 相比，扩增的软骨细胞显示出略高的软骨特异性基因表达，但两种细胞类型释放的蛋白质总体谱非常相似。从定性角度来看，MSCs 似乎分泌相似数量的细胞外基质蛋白（43％对总蛋白质的 45％）和分解代谢剂（9％对 10％），以及更多的合成代谢剂（12％对 7％）与 ACs 相比。一些基质调节因子，如丝氨酸蛋白酶抑制剂、BMP-1 和半乳糖凝集素，仅在 MSC 上清液中检测到。MMPs 和 TIMPs 的定量分析显示，ACs 培养基中 MMP-1、MMP-2、MMP-3 和 MMP-7 的水平明显更高。我们的数据表明，在扩增阶段之后，ACs 和 MSCs 均表达出去分化的表型，彼此相似。在基因表达水平上，ACs 具有更接近天然软骨的表型，而通过观察释放蛋白模式，MSCs 表现出更具合成代谢的特征。我们的数据以及 MSCs 保持其分化潜能更长培养时间的固有能力，将有利于使用这些细胞进行软骨重建。
