【作者】 孟斌；

【导师】 潘新； 李昊；

【作者基本信息】 山东大学 ， 骨科学（专业学位）， 2025， 硕士

【摘要】 研究背景椎间盘退行性变(Intervertebral Disc Degeneration,IVDD)作为各类椎间盘疾病的病理基础,全球约6.37亿人受其引发的腰痛困扰。铁死亡(ferroptosis)作为近年来新发现的一种细胞死亡方式,现有研究表明,铁死亡可通过诱导髓核细胞死亡、促进细胞外基质降解等途径参与IVDD的发生发展。然而,关于铁死亡在IVDD中的具体分子机制,包括其关键调控因子、信号转导通路及其与其他细胞死亡方式的交互作用等科学问题,仍需通过系统的实验研究加以阐明。方法本研究采用RNA测序技术对对照组与IL-1 β处理的IVDD模型组的大鼠腰椎间盘组织进行转录组分析,鉴定差异表达基因(DEGs)。通过生物信息学分析,对获得的DEGs进行基因本体论(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析,以揭示其潜在的生物学功能和信号通路。为探究DEGs与铁死亡相关的差异表达基因(DEFRGs)之间的相互作用关系,利用STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络,并运用Cytoscape软件进行网络拓扑分析,识别关键模块和枢纽基因。为验证PDK4基因的功能,本研究采用siRNA干扰技术在髓核细胞(NPCs)中进行基因敲降实验,并通过CCK-8细胞增殖检测、ELISA细胞因子测定以及铁死亡相关指标分析,系统评估PDK4基因功能缺失对细胞表型的影响。结果使用RNA-seq分析IVDD模型中的DEGs,确定了 487个上调基因和236个下调基因。KEGG通路富集分析结果表明,这些差异表达基因显著富集于多个关键信号通路,包括帕金森病通路、催产素信号通路、钙离子信号通路、AMPK信号通路以及胰高血糖素信号通路。PPI网络和Cytoscape拓扑分析结果显示,在IVDD模型大鼠椎间盘组织中鉴定出的25个铁死亡相关基因中,存在8个关键Hub基因,分别为LDHA、PKM、EP300、EGFR、EGLN1、SCD、PDK4 和 FABP4。qRT-PCR 及 IHC 检测 IVDD 组大鼠8个关键基因的表达水平显示,LDHA、PKM、EGLNI、FABP4和PDK4的mRNA表达较对照组显著上调,EGFR、EP300和SCD表达水平显著下调。通过siRNA特异性沉默PDK4基因表达可促进NPCs增殖,降低IL-18、IL-1β及TNF-α促炎因子水平,并影响铁死亡相关指标:脂质过氧化标志物丙二醛(MDA)水平降低,超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)水平升高,细胞内铁离子含量减少;Western blotting和qRT-PCR结果显示,PDK4沉默抑制ACSL4的表达并上调GPX4和FTH1的表达水平。结论本研究通过RNA测序技术对IVDD大鼠腰椎间盘组织的转录组DEGs进行系统性分析,鉴定出487个上调基因和236个下调基因。我们从这些DEGs中识别出25个DEFRGs,并通过生物信息学分析确定了 8个核心枢纽基因—LDHA、PKM、EP300、EGFR、EGLN1、SCD、PDK4及FABP4。通过siRNA介导的基因敲降实验,我们发现PDK4沉默可促进NPCs增殖、降低炎症水平并抑制细胞铁死亡。PDK4不仅参与调控IVDD病程中的细胞增殖、炎症反应及铁死亡进程,还可能成为连接病理机制与治疗靶点的关键分子。更多还原

【Abstract】 BackgroundsFerroptosis,a newly discovered form of cell death identified in recent years,has been shown in existing studies to participate in the pathogenesis and progression of intervertebral disc degeneration(IVDD)through mechanisms such as inducing nucleus pulposus cell(NPC)death and promoting extracellular matrix(ECM)degradation.However,critical molecular mechanisms underlying ferroptosis in IVDD—including its key regulatory factors,signaling pathways,and interactions with other cell death modalities—remain to be fully elucidated through systematic experimental investigations.MethodsThe present study employed RNA sequencing(RNA-seq)to perform transcriptomic analysis of rat lumbar intervertebral disc tissues from control and IL-1β-treated models of IVDD.Differentially expressed genes(DEGs)were identified through comparative analysis between groups.Subsequently,bioinformatic analyses including Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were conducted to elucidate the underlying biological functions and signaling pathways associated with these DEGs.To explore the regulatory networks involved in ferroptosis,we further screened for differentially expressed ferroptosis-related genes(DEFRGs).Protein-protein interaction(PPI)networks were constructed using the STRING database,followed by topological analysis through Cytoscape software to identify critical modules and hub genes.For functional validation,we performed siRNA-mediated gene knockdown of PDK4 in nucleus pulposus cells(NPCs),followed by systematic phenotypic assessments including CCK-8 cell proliferation assays,ELISA-based cytokine quantification,and analysis of ferroptosis-associated markers.This experimental pipeline aimed to delineate the functional role of PDK4 deficiency in cellular pathophysiology.ResultsRNA-seq analysis of IVDD models identified 487 upregulated genes and 236 downregulated DEGs.KEGG pathway enrichment analysis revealed that these DEGs were significantly enriched in multiple critical signaling pathways,including the Parkinson’s disease pathway,oxytocin signaling pathway,calcium signaling pathway,AMPK signaling pathway,and glucagon signaling pathway.PPI network and Cytoscape topological analysis identified 8 hub genes—LDHA,PKM,EP300,EGFR,EGLN1,SCD,PDK4 and FABP4—among the 25 ferroptosis-related genes detected in IVDD rat disc tissues.qRT-PCR and IHC demonstrated that mRNA expression levels of LDHA,PKM,EGLN1,FABP4 and PDK4 were significantly upregulated in the IVDD group compared to controls,while EGFR,EP300 and SCD expression levels were markedly downregulated.Silencing PDK4 via siRNA promoted NPCs proliferation,reduced pro-inflammatory cytokine levels(IL-18,IL-1β and TNF-α),and modulated ferroptosis-related markers:decreased malondialdehyde(MDA)levels,increased superoxide dismutase(SOD)activity and glutathione(GSH)content and reduced intracellular iron concentration.Western blotting and qRT-PCR further showed that PDK4 silencing inhibited ACSL4 expression while upregulating GPX4 and FTH1.ConclusionsThrough RNA sequencing technology and systematic transcriptomic analysis of intervertebral disc tissues from IVDD rats,this study identified 487 upregulated genes and 236 downregulated DEGs.Among these DEGs,25 DEFRGs were screened,and 8 core hub genes—LDHA,PKM,EP300,EGFR,EGLN1,SCD,PDK4 and FABP4—were determined via bioinformatics analysis.Using siRNA-mediated gene knockdown experiments,we demonstrated that PDK4 silencing promoted NPCs proliferation,reduced inflammatory levels and inhibited cellular ferroptosis.PDK4 not only participates in regulating cell proliferation,inflammatory responses,and ferroptosis during IVDD progression but may also act as a core molecular link between pathological mechanisms and therapeutic targets.更多还原