Tan Sik-Loo, Ahmad Tunku Sara, Ng Wuey-Min, Azlina Amir Abbas, Azhar Mahmood Merican, Selvaratnam Lakshmi, Kamarul Tunku
Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Pantai Valley, Kuala Lumpur, 50603, Malaysia.
School of Medicine & Health Sciences, Monash University, Sunway Campus, Selangor, Malaysia.
PLoS One. 2015 Nov 3;10(11):e0140869. doi: 10.1371/journal.pone.0140869. eCollection 2015.
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.
迄今为止，调节生长因子诱导的间充质干细胞（MSCs）向肌腱细胞分化的分子信号传导机制仍 largely 未知。因此，进行了一项研究，以确定使用生长分化因子 5（GDF5）诱导人骨髓基质细胞（hMSCs）向肌腱细胞分化过程中的全基因组表达谱。对添加了 100 ng/ml GDF5 的 hMSCs 培养物进行了微阵列分析，并与未分化的 hMSCs 和成人肌腱细胞进行了比较。QuantiGene® Plex 检测结果支持了推断的基因表达谱和通路信息的使用及解读。在评估的 27,216 个基因中，有 873 个基因（占人类转录组总数的 3.21%）在向肌腱细胞分化过程中发生了显著变化（校正 p<0.05）。被确定为可能与肌腱细胞分化相关的基因有 ARHGAP29、CCL2、整合素α8 和神经丝中型多肽。这些基因主要与细胞骨架重组（应力纤维形成）信号传导相关。通路分析表明，参与肌腱细胞分化的潜在分子通路有：与细胞骨架重组相关的，即角蛋白丝信号传导和激活素 A 信号传导；与细胞黏附相关的，即趋化因子和黏附信号传导；以及与细胞外基质相关的，即花生四烯酸产生信号传导。使用原子力显微镜和共聚焦激光扫描显微镜进行的进一步研究表明，在 GDF5 诱导的 hMSCs 中明显存在细胞骨架重组，这表明细胞骨架重组信号传导是肌腱细胞分化过程中涉及的一个重要事件。此外，观察到核干细胞表达降低，表明正在进行肌腱细胞分化的 hMSCs 细胞增殖率较低。理解和阐明肌腱细胞分化信号通路对于未来优化用于基于功能性肌腱细胞的治疗和组织工程的肌腱细胞 hMSCs 至关重要。
