摘要

第一章：基于生物信息学与机器学习确定免疫相关基因在类风湿性关节炎并发动脉粥样硬化诊断中的应用研究

背景：类风湿性关节炎与动脉粥样硬化的共病机制尚不明确。近年研究表明，类风湿性关节炎患者合并动脉粥样硬化发病率显著升高，其免疫微环境紊乱可能是关键驱动因素。本研究旨在通过生物信息学分析，筛选类风湿性关节炎与动脉粥样硬化共同的免疫调控关键基因，并构建诊断模型。

方法：从GEO数据库获取动脉粥样硬化数据集GSE100927与类风湿性关节炎GSE55457转录组数据集，采用Limma算法筛选差异表达基因，通过WGCNA构建共表达网络识别核心模块基因，并整合差异表达基因与模块基因的共同基因，利用STRING数据库构建蛋白质互作网络，结合Cytoscape的MCODE插件识别枢纽基因。进一步采用LASSO回归与随机森林算法筛选特征基因，通过列线图可视化诊断模型，并利用独立数据集GSE57691，GSE55235进行外部验证。采用CIBERSORT算法解析动脉粥样硬化患者的免疫细胞浸润特征。

结果：通过生物信息学分析，本研究系统揭示了类风湿性关节炎与动脉粥样硬化共同的免疫调控机制：首先利用Limma算法筛选获得动脉粥样硬化相关差异表达基因5322个，结合类风湿性关节炎患者的2705个因与WGCNA鉴定的206个模块基因，取交集3个免疫相关交集基因；进一步通构建蛋白互作网络。并利用LASSO回归分析与随机森林联合筛选，最终锁定NFIL3、EED、GRK2、MAP3K11、RMI1和TPST1等6个枢纽基因。利用6个枢纽基因构建列线图和进行诊断效能评估，显示出较大的诊断价值，免疫浸润分析进一步发现患者免疫细胞变化情况。

结论：本研究通过生物信息学分析鉴定出NFIL3、EED、GRK2、MAP3K11、RMI1和TPST1等6个免疫调控枢纽基因，并基于机器学习构建出列线图诊断模型，并表现出优异效能，为类风湿性关节炎患者合并动脉粥样硬化的早期筛查提供了新型生物标志物组合。

第二章：利用生物信息学对负载磁性纳米颗粒的PLGA支架联合静磁场对骨缺损修复作用的机制探究

目的：骨缺损修复一直是骨科和组织工程领域的重要研究方向之一。骨缺损可能由创伤、感染、肿瘤切除或先天性畸形引起，严重影响患者的生活质量。传统的治疗手段主要包括自体骨移植和异体骨移植，但这些方法面临诸多局限性：自体骨移植易导致供区并发症且受限于供区骨量，异体骨移植则可能引发免疫排斥反应和感染风险。此外，传统移植材料在修复复杂骨缺损方面表现不足，难以满足临床需求。因此，开发更高效的骨再生策略是当前研究的热点和难点。血管新生在骨再生过程中发挥了不可或缺的作用。骨组织不仅需要充足的矿化基质，还需建立完善的血管网络以提供营养和氧气，并清除代谢废物。然而，现有许多骨修复材料仅注重成骨特性，而忽视了血管新生的重要性。研究表明，增强骨缺损区的血管生成可显著提高骨再生的效率和质量。因此，设计兼具促成骨与促血管特性的骨修复材料对组织工程骨再生具有重要意义。近年来，3D打印技术的快速发展为骨修复材料的研发带来了全新机遇。通过3D打印，可以制备出具有高度复杂结构和定制化设计的支架材料，这些支架能够更好地匹配骨缺损区域的形状并提供理想的细胞附着和组织生长微环境。此外，磁性纳米材料（如Fe₃O₄、γ-Fe₂O₃）因其优异的生物相容性和在外部磁场下的特殊性能，在促进骨再生方面展现了巨大潜力。研究表明，磁性纳米材料与静磁场（static magnetic field,SMF）联合使用能够显著增强骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMSCs）的成骨分化，同时对血管新生也具有促进作用。因此，将MNPs与3D打印支架结合，并在SMF条件下使用，为骨再生研究提供了一种新颖且有前景的策略。本研究旨在探讨负载Fe₂O₃MNPs的3D打印聚乳酸-羟基乙酸共聚物（Poly lactic-co-glycolic acid, PLGA）支架结合电子扫描显微镜（Scanning Electron Microscope，SMF）在骨再生中的作用机制，重点分析其在成骨和血管生成方面的表现。通过体外细胞实验和体内动物模型验证，深入研究支架对BMSCs和人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cell，HUVEC）功能的调控作用，并解析相关分子通路在成骨与成血管过程中的具体贡献，为未来骨再生材料的设计和应用提供理论依据。

方法：使用PLGA作为基材，加载不同含量的Fe₂O₃纳米颗粒（0%、10%、20%），通过3D打印技术制备多孔支架材料。支架的物理化学性能（孔隙率、力学性能、磁性强度）通过扫描电子显微镜（scanning electron microscopy，SEM）和材料试验机进行表征。前期研究已经证实负载磁性纳米颗粒的PLGA支架可以通过调节成骨成血管促进骨再生，本次研究进一步通过蛋白质组学测序和生物信息学分析，探讨PLGA支架促进成骨与成血管的潜在分子通路。重点关注PI3K/AKT信号通路和NF-κB信号通路，结合Western blotting进一步验证关键蛋白的表达和活性变化。

      结果：结果表明，蛋白组学测序结果以及生物信息学分析发现，其成骨促进作用与PI3K/AKT通路有关，磷脂酰肌醇-3激酶 (Phosphatidylinositol-3 kinase，PI3K) 的激活可促进蛋白激酶B（Protein Kinase B，AKT）的激活，进而诱导磷酸化的糖原合酶激酶-3β(Glycogen Synthase Kinase-3β,GSK-3β)失活，从而抑制β-catenin 的磷酸化，减少其泛素化降解。其中，GSK-3β的总表达水平没有显著差异，而Ser9位的GSK-3β表达水平显著下降。同时，CRYAB表达显著上调，这对于通过降低泛素化来稳定β-catenin至关重要。因此，αB-晶状体蛋白（Alpha-B Crystallin，CRYAB）表达的上调促进了β-catenin在核内的聚集，β-catenin是PI3K/AKT信号转导的核心元件，已被证明对人BMSCs成骨分化很重要，而磷酸化的GSK-3β的失活可抑制β-catenin 磷酸化并促进其进入细胞核，因此存在细胞核内磷酸化的βcatenin下降。最后，βcatenin在细胞核内启动下游成骨相关基因的转录。而成血管作用则与NF-κB通路有关，NF-kB 的亚基与HIF-1α启动子-197/188 bp处的kB结合位点相互作用，从而诱导HIF-1α表达，而HIF-1α与VEGF启动子中的HRE结合则进一步诱导VEGF生成，从而促进血管生成。我们进一步通过western blotting证实相关通路蛋白的表达来促进成骨和血管生成。

结论：PI3K/AKT通路通过调控β-catenin稳定性促进成骨分化，而NF-κB通路通过HIF-1α和VEGF的调控促进血管生成。本研究不仅揭示了MNPs与静磁场在骨再生中的协同作用，还为组织工程骨修复材料的设计提供了全新思路。本研究揭示了磁性纳米材料结合SMF对成骨和血管生成的作用机制，为组织工程领域提供了新的理论依据。负载MNPs的PLGA支架具有良好的生物相容性和成骨促血管性能，可为临床骨缺损修复提供新选择，尤其是在复杂骨缺损的修复中具有重要应用价值。将磁性纳米材料与3D打印技术相结合，创新性地实现了支架材料的定制化设计与功能化优化，为骨再生材料的研发开辟了新方向。本研究为进一步开发多功能化、智能化的骨修复支架提供了研究基础，同时为探索其他组织工程材料的设计与优化提供了参考。

Abstract

Part1. Identification of Immune-Related Genes in Diagnosing Atherosclerosis with Rheumatoid arthritis Through Bioinformatics Analysis and Machine Learning.

Objective: Increasing evidence has proven that rheumatoid arthritis (RA) can aggravate atherosclerosis (AS), and we aimed to explore potential diagnostic genes for patients with AS and RA.

Methods: We obtained the data from public databases, including Gene Expression Omnibus (GEO) and STRING, and obtained the differentially expressed genes (DEGs) and module genes with Limma and weighted gene co-expression network analysis (WGCNA). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, the protein–protein interaction (PPI) network, and machine learning algorithms [least absolute shrinkage and selection operator (LASSO) regression and random forest] were performed to explore the immune-related hub genes. We used a nomogram and receiver operating characteristic (ROC) curve to assess the diagnostic efficacy, which has been validated with GSE55235 and GSE57691. Finally, immune infiltration was developed in AS.

Results: The AS dataset included 5,322 DEGs, while there were 2705 DEGs and 206 module genes in RA. The intersection of DEGs for AS and crucial genes for RA was 53, which were involved in immunity. After the PPI network and machine learning construction, six hub genes were used for the construction of a nomogram and for diagnostic efficacy assessment, which showed great diagnostic value (area under the curve from 0.723 to 1). Immune infiltration also revealed the disorder of immunocytes.

Conclusion: Six immune-related hub genes (NFIL3, EED, GRK2, MAP3K11, RMI1, and TPST1) were recognized, and the nomogram was developed for AS with RA diagnosis.

Part2. Mechanistic Investigation of Bone Defect Repair via Magnetic Nanoparticle-Loaded PLGA Scaffolds Combined with Static Magnetic Field: A Bioinformatics-Based Study.

Objective: Bone defect repair has always been one of the important research directions in orthopedics and tissue engineering. Bone defects may be caused by trauma, infection, tumor resection, or congenital deformity, which severely impacts the patient 's quality of life. Traditional treatments mainly include autologous bone graft and allogeneic bone graft, but these methods face many limitations: autologous bone graft easily leads to donor site complications and is limited by donor site bone mass, and allogeneic bone graft may trigger immune rejection and infection risk. In addition, traditional graft materials have shown insufficient performance in repairing complex bone defects and are difficult to meet clinical needs. Therefore, the development of more efficient bone regeneration strategies is a hot and difficult topic in current research. Angiogenesis plays an integral role in bone regeneration. Bone tissue requires not only sufficient mineralized matrix, but also a well-established vascular network to provide nutrients and oxygen and remove metabolic waste. However, many existing bone repair materials only focus on osteogenic properties and ignore the importance of angiogenesis. It has been shown that enhancing angiogenesis in the bone defect area significantly increases the efficiency and quality of bone regeneration. Therefore, it is important to design bone repair materials that have both bone-promoting and vascularizing properties for tissue-engineered bone regeneration. In recent years, the rapid development of 3D printing technology has brought brand-new opportunities for the research and development of bone repair materials. By 3D printing, scaffold materials with highly complex structures and customized designs can be prepared that better match the shape of the bone defect area and provide an ideal cell attachment and tissue growth microenvironment. In addition, magnetic nanomaterials (such as Fe O, γ -Fe O) have shown great potential in promoting bone regeneration because of their excellent biocompatibility and special properties under external magnetic fields. Studies have shown that magnetic nanomaterials combined with static magnetic field (SMF) can significantly enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), while also promoting angiogenesis. Therefore, combining magnetic nanoparticles with 3D printed scaffolds and using them under SMF conditions provides a novel and promising strategy for bone regeneration studies. The aim of this study was to investigate the mechanism of 3D printed PLGA（Poly lactic-co-glycolic acid） scaffolds loaded with Fe₂O₃ magnetic nanoparticles combined with SMF in bone regeneration, focusing on their performance in osteogenesis and angiogenesis. Through in vitro cell experiments and in vivo animal models, the regulation of BMSCs and HUVECs（Human Umbilical Vein Endothelial Cell） function by scaffolds was studied in depth, and the specific contribution of related molecular pathways in the process of osteogenesis and angiogenesis was analyzed to provide a theoretical basis for the design and application of future bone regeneration materials.

Methods: Porous scaffolds were prepared by 3D printing technique using polylactide-glycolic acid copolymer (PLGA) as a substrate loaded with different contents of Fe₂O₃ nanoparticles (0%, 10%, 20%). The potential molecular pathways of PLGA scaffolds promoting osteogenesis and angiogenesis were investigated by proteomic sequencing and bioinformatics analysis. Focus on PI3K/AKT signaling pathway and NF-κB signaling pathway, combined with Western blotting to further verify the expression and activity changes of key proteins.

Results: Proteomics sequencing results as well as bioinformatics analysis revealed that its osteogenic promoting effect was related to the PI3K/AKT pathway, and the activation of PI3K could promote the activation of AKT, which in turn induced the inactivation of phosphorylated GSK-3β, thereby inhibiting the phosphorylation of β-catenin and reducing its ubiquitination and degradation. Among them, the total expression level of GSK-3β was not significantly different, while the GSK-3β expression level at Ser9 position was significantly decreased. Meanwhile, CRYAB expression was significantly upregulated, which is essential for stabilization of β-catenin by decreasing ubiquitination. Thus, upregulation of CRYAB expression promotes nuclear accumulation of β-catenin, a core element of PI3K/AKT signaling that has been shown to be important for osteogenic differentiation in human BMSCs, whereas inactivation of phosphorylated GSK-3β inhibits β-catenin phosphorylation and promotes its entry into the nucleus, so there is a decrease in phosphorylated β-catenin in the nucleus. Finally, β-catenin initiates transcription of downstream osteogenesis-related genes in the nucleus. Angiogenesis, on the other hand, is associated with the NF-κB pathway, and subunits of NF-kB interact with the kB binding site at − 197/188 bp of the HIF-1α promoter to induce HIF-1α expression, while HIF-1α binding to HRE in the VEGF promoter further induces VEGF production, thereby promoting angiogenesis. We further confirmed the expression of related pathway proteins by western blotting to promote osteogenesis and angiogenesis.

Conclusion: 3D printed PLGA scaffolds loaded with Fe O nanoparticles can significantly promote osteogenesis and angiogenesis under SMF.PI3K/AKT pathway promotes osteogenic differentiation by regulating β-catenin stability, while NF-κB pathway promotes angiogenesis through the regulation of HIF-1α and VEGF. This study not only reveals the synergistic effect of magnetic nanoparticles and static magnetic field in bone regeneration, but also provides a brand-new idea for the design of tissue-engineered bone repair materials. This study reveals the mechanism of magnetic nanomaterial combined with SMF on osteogenesis and angiogenesis, which provides a new theoretical basis for the field of tissue engineering. PLGA scaffolds loaded with magnetic nanoparticles have good biocompatibility and osteogenic and provascular properties, which can provide new options for clinical bone defect repair, especially in the repair of complex bone defects. Combining magnetic nanomaterials with 3D printing technology, customized design and functionalization optimization of scaffold materials have been innovatively realized, which opens up a new direction for the research and development of bone regeneration materials. This study provides a research basis for further development of multifunctional and intelligent bone repair scaffolds, while providing a reference for exploring the design and optimization of other tissue engineering materials.