【作者】 张鹏；

【导师】 朱晨；

【作者基本信息】 安徽医科大学 ， 骨科学， 2025， 硕士

【摘要】 目的与背景:骨科内植物相关感染是造成骨科手术失败的最常见的原因和最严重的术后并发症。造成难治性内植物感染主要原因在于细菌定植内植物表面形成了致密生物膜结构。细菌生物膜是由细菌分泌胞外DNA,蛋白质,胞外基质等组成的复杂聚集体,具有低氧,低p H等特点,能够限制抗生素渗透,抑制免疫系统侵袭。同时,生物膜内部细菌选择性耐药性繁殖,变异,进一步加大临床上抗感染治疗难度。复杂的细菌生物膜导致感染周围抑制性免疫微环境,特别是感染组织周围巨噬细胞在面临逸散细菌时形成的胞外陷阱结构,造成感染迁延不愈,导致彻底的清除生物膜感染愈发困难。通过清除生物膜感染和重塑感染周围免疫抑制微环境,特别是抑制巨噬细胞胞外陷阱结构防止逃逸的细菌再次形成生物膜,能够有效解决临床上难治性内植物细菌生物膜感染。方法:在此,我们利用类超氧化物歧化酶（SOD）MOF-818和掺铁（Fe）沸石咪唑框架（Fe-ZIF）,形成双层MOF纳米酶,加入氧气吸附载体全氟己烷（PFH）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）,形成智能响应型抗菌纳米酶MPFN-O₂。通过对MPFN-O₂的体外表征实验,体外抗菌实验,抑制巨噬细胞胞外陷阱等免疫实验和体内小鼠皮下内植物感染模型,验证其能够彻底清除细菌生物膜感染和抑制巨噬细胞胞外陷阱结构减少细菌逃逸重塑免疫微环境。结果:（1）成功制备抗菌纳米酶MPFN-O₂,通过材料表征实验进行验证。（2）MPFN-O₂在酸性生物膜环境下产生有毒的羟基自由基（·OH）,破坏细菌生物膜。局部释放的铁离子破坏细菌DNA,进一步清除细菌。（3）MPFN-O₂抑制感染周围巨噬细胞胞外陷阱形成,增强巨噬细胞杀菌能力,募集循环中性粒细胞彻底清除细菌生物膜感染。（4）构建内植物感染小鼠模型,验证MPFN-O₂高效的抗菌能力。结论:抗菌纳米酶MPFN-O₂能够破坏细菌生物膜,清除细菌感染,抑制内植物周围组织巨噬细胞胞外陷阱结构减少细菌逃逸,增强免疫系统杀菌能力,重塑免疫微环境,为临床上彻底清除内植物细菌生物膜感染提供了新的思路。更多还原

【Abstract】 Objective and Background:Implant-associated infections are the most common cause of surgical failure and the most severe postoperative complication in orthopedic surgery.The primary cause of refractory implant infections is the formation of the dense biofilm structure by bacteria that colonize the surface of the implant.Bacterial biofilms are complex aggregates composed of extracellular DNA,proteins,and extracellular matrix secreted by bacteria.These biofilms have characteristics such as low oxygen and low p H,which limit the penetration of antibiotics and suppress the immune system to eradicate.Additionally,bacteria within the biofilm exhibit selective antibiotic resistance,proliferate,and mutate,further inducing complicate clinical treatment of infections.Furthermore,the complex bacterial biofilm creates an immunosuppressive microenvironment around the infection,especially the extracellular traps structures formed by macrophages in response to escaping bacteria,which leading to persistent infections.This makes it increasingly difficult to completely eradicate biofilm-associated infections.Therefore,effectively resolving refractory implant-associated bacterial biofilm infections requires clearing the biofilm and remodeling the immunosuppressive microenvironment,particularly inhibiting the macrophage extracellular trap structures.Methods:In this study,we utilized superoxide dismutase（SOD）-mimicking MOF-818and iron-doped zeolitic imidazolate frameworks（Fe-ZIF）to form a dual MOF nanoenzyme.Oxygen-adsorbing perfluorohexane（PFH）and nicotinamide adenine dinucleotide phosphate（NADPH）were incorporated to create an intelligent response antimicrobial nanoenzyme（MPFN-O₂）.The in vitro characterization experiments,antimicrobial assays,immune experiments to inhibit macrophage extracellular trap formation,and in vivo mouse subcutaneous implant infection model all verified that MPFN-O₂ can effectively eliminate bacterial biofilm infections and inhibit macrophage extracellular trap structures,reducing bacterial escape and remodeling the immune microenvironment.Results:（1）The MPFN-O₂ was successfully synthesized,and its material characterization was validated through experiments;（2）MPFN-O₂ generates toxic hydroxyl radicals（·OH）in the acidic biofilm environment,which disrupt bacterial biofilms.Locally released iron ions damage bacterial DNA,further eliminating bacteria;（3）MPFN-O₂ inhibits the formation of macrophage extracellular traps around the infection,enhances the bactericidal capacity of macrophages,and recruits circulating neutrophils to thoroughly eliminate bacterial biofilm infections;（4）A mouse model of implant infection was constructed to validate the efficient antimicrobial activity of MPFN-O₂.Conclusion:The antimicrobial nanoenzyme MPFN-O₂ have ability in disrupting bacterial biofilms,clearing bacterial infections and inhibiting the formation of macrophage extracellular trap structures around the implant.Additionally,it can reduce bacterial escape,enhance the bactericidal capacity of the immune system,and remodel the immune microenvironment.This provides a novel solution for the complete eradication of implant-associated bacterial biofilm infections in clinical practice.更多还原