中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (36): 7783-7789.doi: 10.12307/2025.562

• 干细胞外泌体 Stem cell exosomes • 上一篇    下一篇

骨关节炎关节液外泌体中软骨退变相关基因筛选及细胞学验证

马维邦1,徐  哲1,2,喻  乔2,3,欧阳东1,张如国1,罗  伟1,谢阳江1,刘  琛1   

  1. 1贵航贵阳医院骨科,贵州省贵阳市   550005;2 贵州医科大学,贵州省贵阳市   550042;3贵黔国际总医院骨科,贵州省贵阳市   550018
  • 收稿日期:2024-09-23 接受日期:2024-11-14 出版日期:2025-12-28 发布日期:2025-03-10
  • 通讯作者: 刘琛,学士,主治医师,贵航贵阳医院骨科,贵州省贵阳市 550005
  • 作者简介:马维邦,男,1991年生,山东省烟台市人,汉族,2021年贵州医科大学毕业,硕士,主治医师,主要从事骨与关节的临床与基础研究。
  • 基金资助:
    通用医疗科研基金项目(TYYLKYJJ-2023-032),项目负责人:马维邦;贵航贵阳医院科研基金项目(GHGYYY—KYLX-2022-14),项目负责人:马维邦;贵州省基础研究计划(黔科和基础-ZK [2023]一般532),项目负责人:徐哲

Screening and cytological validation of cartilage degeneration-related genes in exosomes from osteoarthritis synovial fluid

Ma Weibang1, Xu Zhe1, 2, Yu Qiao2, 3, Ouyang Dong1, Zhang Ruguo1, Luo Wei1, Xie Yangjiang1, Liu Chen1   

  1. 1Department of Orthopedics, Guihang Guiyang Hospital, Guiyang 550005, Guizhou Province, China; 2Guizhou Medical University, Guiyang 550042, Guizhou Province, China; 3Department of Orthopedics, Guiqian International General Hospital, Guiyang 550018, Guizhou Province, China
  • Received:2024-09-23 Accepted:2024-11-14 Online:2025-12-28 Published:2025-03-10
  • Contact: Liu Chen, Attending physician, Department of Orthopedics, Guihang Guiyang Hospital, Guiyang 550005, Guizhou Province, China
  • About author:Ma Weibang, MS, Attending physician, Department of Orthopedics, Guihang Guiyang Hospital, Guiyang 550005, Guizhou Province, China
  • Supported by:
    General Medical Research Fund Project, No. TYYLKYJJ-2023-032 (to MWB); Guihang Guiyang Hospital Research Fund Project, No. GHGYYY—KYLX-2022-14 (to MWB); Guizhou Basic Research Program, No. Qian Kehe Jichu-ZK [2023] General 532 (to XZ)

摘要:

文题释义:

外泌体:是一种广泛存在于机体全身、由脂质双分子层包裹的微小囊泡,直径一般为40-100 nm,其内容物含有多种核酸如DNA、mRNA、miRNA以及蛋白质,可以发挥细胞间物质运输和信息传递的作用。
软骨退行性变:指四肢关节因创伤或慢性劳损而产生的关节软骨变性退化、增殖性新骨形成等病理变化。临床X射线片表现为关节间隙变窄、关节面增生硬化、骨赘形成以及韧带钙化。

摘要
背景:已有研究证实外泌体与骨关节炎软骨退变密切相关。然而,外泌体来源基因在骨关节炎软骨退变中的作用及机制尚未被完全阐明。
目的:通过生物信息技术筛选骨关节炎患者关节液外泌体中与软骨退变相关的基因,确定其生物学功能和信号通路,进而为延缓骨关节炎软骨退变提供新的治疗靶点。
方法:首先,从基因表达综合(gene expression omnibus,GEO)数据库中下载骨关节炎相关外泌体数据集GSE185059和软骨退变数据集GSE114007,筛选外泌体来源软骨退变相关基因。然后对筛选的外泌体来源软骨退变相关基因进行GO功能及KEGG通路富集分析,绘制蛋白质-蛋白质相互作用网络以及IPA分析,筛选获得外泌体来源软骨退变相关的关键基因。最后,采用qRT-PCR实验验证骨关节炎软骨组织以及白细胞介素1β刺激软骨细胞模型中关键基因的表达情况。
结果与结论:① GSE185059数据集中共有831个差异表达基因,GSE114007数据集中共有5 323个差异表达基因。两者取交集后共筛选获得94个外泌体来源软骨退变相关基因,其中51个基因表达下调,43个基因表达上调。②GO功能富集分析表明,上调的基因主要参与细胞-细胞黏附的正向调节、T细胞活化的正调控以及慢性炎症反应等生物学过程,下调的基因主要参与细胞聚集、软骨分化发育以及骨骼系统形态发生等生物学过程。③KEGG通路富集分析发现,外泌体来源软骨退变相关基因主要参与富集色氨酸代谢、维生素B6代谢以及白细胞跨内皮迁移等信号通路。④构建的蛋白质-蛋白质相互作用网络证实,外泌体来源软骨退变相关基因之间存在多个相互作用关系。结合CytoHubba插件中的5种算法进一步筛选获得了4个关键外泌体来源软骨退变相关基因,分别为THY1、CYP1A1、NFKB2、COL6A3。⑤qRT-PCR结果证实,相比于正常软骨组织,骨关节炎软骨组织中THY1、COL6A3表达升高,而CYP1A1、NFKB2表达降低;同样,相比于未刺激组,白细胞介素1β刺激组软骨细胞中THY1、COL6A3表达升高,而CYP1A1、NFKB2表达降低。⑥以上结果表明,THY1、CYP1A1、NFKB2和COL6A3是骨关节炎关节液外泌体中与软骨退变相关的基因,可能通过调控蛋白酪氨酸激酶活性以及脂质代谢等生物学过程以及核因子κB信号通路、黏着斑信号通路等参与骨关节炎疾病进程。然而,这些关键基因在软骨退变中的具体调控作用和分子机制有待进一步实验验证。

关键词: 骨关节炎, 关节液, 外泌体, 软骨退变, 生信分析, 细胞学验证, 软骨细胞, 基因表达, 工程化外泌体

Abstract: BACKGROUND: Exosomes have been confirmed to be closely related to cartilage degeneration in osteoarthritis. However, the role and mechanism of exosome-derived genes in cartilage degeneration of osteoarthritis have not been fully elucidated.
OBJECTIVE: Bioinformatics analyses were used to screen the genes related to cartilage degeneration in the synovial exosomes of patients with osteoarthritis, and to determine their biological functions and signaling pathways in order to provide new therapeutic targets for delaying cartilage degeneration in osteoarthritis.
METHODS: Firstly, osteoarthritis-related exosome dataset GSE185059 and cartilage degeneration dataset GSE114007 were downloaded from Gene Expression Omnibus (GEO) database to screen exosome-derived cartilage degeneration related genes. GO functional and KEGG pathway enrichment analyses were performed based on the screened exosome-derived cartilage degeneration related genes. Protein-protein interaction network was drawn and Ingenuity Pathway Analysis (IPA) was conducted to screen and obtain key exosome-derived cartilage degeneration-related genes. Finally, qRT-PCR was used to verify the expression of key genes in osteoarthritis cartilage tissue and interleukin-1β stimulated chondrocyte models. 
RESULTS AND CONCLUSION: (1) There were 831 differentially expressed genes in the GSE185059 dataset and 5 323 differentially expressed genes in the GSE114007 dataset. A total of 94 exosome-derived cartilage degeneration related genes were screened after the intersection of these differentially expressed genes, of which 51 genes were down-regulated and 43 genes were up-regulated. (2) GO functional enrichment analysis showed that the up-regulated genes were mainly involved in the positive regulation of cell-cell adhesion, the positive regulation of T cell activation, and chronic inflammatory response, while the down-regulated genes were mainly involved in biological processes such as cell aggregation, cartilage differentiation and development, and skeletal system morphogenesis. (3) KEGG pathway enrichment analysis showed that exosome-derived cartilage degeneration-related genes were mainly involved in tryptophan enrichment metabolism, vitamin B6 metabolism, and leukocyte transendothelial migration. (4) The constructed protein-protein interaction network confirmed the existence of multiple interaction relationships among exosome-derived cartilage degeneration-related genes. Combined with five algorithms in CytoHubba software, four key exosome-derived cartilage degeneration-related genes were further screened, namely THY1, CYP1A1, NFKB2, and COL6A3. (5) The results of qRT-PCR showed that compared with normal cartilage, the expressions of THY1 and COL6A3 in osteoarthritic cartilage were increased, while the expression of CYP1A1 and NFKB2 was decreased. Similarly, compared with the unstimulated group, the expression of THY1 and COL6A3 in the interleukin-1β induced chondrocytes was upregulated, while the expression of CYP1A1 and NFKB2 was downregulated. (6) These results indicate that THY1, CYP1A1, NFKB2, and COL6A3 are genes related to cartilage degeneration in the exosomes of synovial fluid of patients with osteoarthritis, and may participate in the pathogenesis of osteoarthritis by regulating biological processes such as protein tyrosine kinase activity and lipid metabolism, as well as nuclear factor-κB signaling pathway and focal adhesion signaling pathway. However, the specific regulatory roles and molecular mechanisms of these key genes in cartilage degeneration need to be further verified by experiments.


Key words: ">osteoarthritis, synovial fluid, exosome, cartilage degeneration, bioinformatics analysis, cytological verification, chondrocyte, gene expression, engineered exosomes

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