Cheng Shi, Li Xiaochuan, Lin Linghan, Jia Zhiwei, Zhao Yachao, Wang Deli, Ruan Dike, Zhang Yu
Department of Orthopedics, Guangdong General Hospital, Guangdong Academy of Medical Science, South China University of Technology, Guangzhou 510080, China.
Department of Orthopedic Surgery, Gaozhou People's Hospital, Guangdong 525200, China.
Stem Cells Int. 2019 Jul 10;2019:2785207. doi: 10.1155/2019/2785207. eCollection 2019.
Nucleus pulposus cells (NPCs) play a vital role in maintaining the homeostasis of the intervertebral disc (IVD). Previous studies have discovered that NPCs exhibited malfunction due to cellular senescence during disc aging and degeneration; this might be one of the key factors of IVD degeneration. Thus, we conducted this study in order to investigate the altered biofunction and the underlying genes and pathways of senescent NPCs. We isolated and identified NPCs from the tail discs of young (2 months) and old (24 months) SD rats and confirmed the senescent phenotype through SA--gal staining. CCK-8 assay, transwell assay, and cell scratch assay were adopted to detect the proliferous and migratory ability of two groups. Then, a rat Gene Chip Clariom™ S array was used to detect differentially expressed genes (DEGs). After rigorous bioinformatics analysis of the raw data, totally, 1038 differentially expressed genes with a fold change > 1.5 were identified out of 23189 probes. Among them, 617 were upregulated and 421 were downregulated. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted and revealed numerous number of enriched GO terms and signaling pathways associated with senescence of NPCs. A protein-protein interaction (PPI) network of the DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software. Module analysis was conducted for the PPI network using the MCODE plugin in Cytoscape. Hub genes were identified by the CytoHubba plugin in Cytoscape. Derived 5 hub genes and most significantly up- or downregulated genes were further verified by real-time PCR. The present study investigated underlying mechanisms in the senescence of NPCs on a genome-wide scale. The illumination of molecular mechanisms of NPCs senescence may assist the development of novel biological methods to treat degenerative disc diseases.
髓核细胞（NPCs）在维持椎间盘（IVD）的内环境稳定中起着至关重要的作用。先前的研究发现，在椎间盘老化和退变过程中，NPCs因细胞衰老而出现功能异常；这可能是IVD退变的关键因素之一。因此，我们开展了本研究，以探究衰老NPCs的生物功能改变以及潜在的基因和信号通路。我们从年轻（2个月）和年老（24个月）的SD大鼠尾椎间盘中分离并鉴定了NPCs，并通过SA-β-半乳糖苷酶染色确认了衰老表型。采用CCK-8法、Transwell法和细胞划痕试验检测两组细胞的增殖和迁移能力。然后，使用大鼠基因芯片Clariom™ S阵列检测差异表达基因（DEGs）。对原始数据进行严格的生物信息学分析后，在23189个探针中总共鉴定出1038个差异表达基因，其倍数变化>1.5。其中，617个基因上调，421个基因下调。此外，进行了基因本体（GO）和京都基因与基因组百科全书（KEGG）信号通路分析，揭示了大量与NPCs衰老相关的富集GO术语和信号通路。使用检索相互作用基因的搜索工具（STRING）数据库和Cytoscape软件构建了DEGs的蛋白质-蛋白质相互作用（PPI）网络。使用Cytoscape中的MCODE插件对PPI网络进行模块分析。通过Cytoscape中的CytoHubba插件鉴定枢纽基因。通过实时PCR进一步验证了衍生的5个枢纽基因以及上调或下调最显著的基因。本研究在全基因组范围内探究了NPCs衰老的潜在机制。对NPCs衰老分子机制的阐明可能有助于开发治疗椎间盘退变疾病的新型生物学方法。
