Peffers Mandy J, Collins John, Loughlin John, Proctor Carole, Clegg Peter D
Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Chester High Road, Neston, CH64 7TE, UK.
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, The University of Liverpool, Leahurst, Neston, CH64 7TE, UK.
Stem Cell Res Ther. 2016 Sep 14;7(1):133. doi: 10.1186/s13287-016-0384-2.
Mesenchymal stem cells (MSCs) have prospective applications in regenerative medicine and tissue engineering but to what extent phenotype and differentiation capacity alter with ageing is uncertain. Consequently, any loss in functionality with age would have profound consequences for the maintenance of tissue viability and the quality of tissues. Proteomics enables the set of proteins responsible for a particular cell phenotype to be identified, as well as enabling insights into mechanisms responsible for age-related alterations in musculoskeletal tissues. Few proteomic studies have been undertaken regarding age-related effects on tissue engineered into cartilage and bone, and none for tendon. This study provides a proteome inventory for chondrogenic, osteogenic and tenogenic constructs synthesised from human MSCs, and elucidates proteomic alterations as a consequence of donor age.
Human bone-marrow derived MSCs from young (n = 4, 21.8 years ± 2.4SD) and old (n = 4, 65.5 years ± 8.3SD) donors were used to make chondrogenic, osteogenic and tenogenic tissue-engineered constructs. We utilised an analytical method relying on extracted peptide intensities as a label-free approach for peptide quantitation by liquid chromatography-mass spectrometry. Results were validated using western blotting.
We identified proteins that were differentially expressed with ageing; 128 proteins in chondrogenic constructs, 207 in tenogenic constructs and four in osteogenic constructs. Differentially regulated proteins were subjected to bioinformatic analysis to ascertain their molecular functions and the signalling pathways. For all construct types, age-affected proteins were involved in altered cell survival and death, and antioxidant and cytoskeletal changes. Energy and protein metabolism were the principle pathways affected in tenogenic constructs, whereas lipid metabolism was strongly affected in chondrogenic constructs and mitochondrial dysfunction in osteogenic constructs.
Our results imply that further work on MSC-based therapeutics for the older population needs to focus on oxidative stress protection. The differentially regulated proteome characterised by this study can potentially guide translational research specifically aimed at effective clinical interventions.
间充质干细胞（MSCs）在再生医学和组织工程领域具有潜在应用价值，但随着衰老其表型和分化能力的改变程度尚不确定。因此，功能随年龄的任何丧失都将对组织活力的维持和组织质量产生深远影响。蛋白质组学能够识别负责特定细胞表型的蛋白质组，还能深入了解肌肉骨骼组织中与年龄相关改变的机制。关于年龄对软骨和骨组织工程的影响，很少有蛋白质组学研究，而对于肌腱则尚无此类研究。本研究提供了由人MSCs合成的软骨生成、骨生成和肌腱生成构建体的蛋白质组清单，并阐明了由于供体年龄导致的蛋白质组学改变。
使用来自年轻供体（n = 4，21.8岁±2.4标准差）和老年供体（n = 4，65.5岁±8.3标准差）的人骨髓来源的MSCs制作软骨生成、骨生成和肌腱生成的组织工程构建体。我们采用一种基于提取肽强度的分析方法，作为通过液相色谱 - 质谱法进行肽定量的无标记方法。结果通过蛋白质印迹法进行验证。
我们鉴定出了随衰老差异表达的蛋白质；软骨生成构建体中有128种蛋白质，肌腱生成构建体中有207种，骨生成构建体中有4种。对差异调节的蛋白质进行生物信息学分析，以确定其分子功能和信号通路。对于所有构建体类型，受年龄影响的蛋白质都涉及细胞存活和死亡的改变以及抗氧化和细胞骨架变化。能量和蛋白质代谢是肌腱生成构建体中受影响的主要途径，而脂质代谢在软骨生成构建体中受到强烈影响，线粒体功能障碍在骨生成构建体中受到影响。
我们的结果表明，针对老年人群基于MSCs的治疗方法的进一步研究需要关注氧化应激保护。本研究表征的差异调节蛋白质组可能潜在地指导专门针对有效临床干预的转化研究。
