基于细菌生物膜反应器构建大肠埃希菌生物膜感染力学生物学机制之研究

Although the fact that bacterial biofilm formation is the key pathogenesis of biomaterial-associated infection has been recognized, the formative mechanism of bacterial biofilm remains unclear. Under the effect of stress stimulation, all organs, tissues and cells of living organism are in the state of physiological strain, and present complex biological consequences. Meanwhile, the mechanical loading have a significant impact on the injury or disease of organism. Our previous studies have confirmed the hydrostatic pressure stimulation could promote the bacterial biofilm formation of Escherichia coli on the carrier surface and shown time-volume relationship. Accordingly, if the dynamic mechanical stimulation of the bladder urine flow could regulate and control the bacterial biofilm formation of Escherichia coli on the indwelling catheter surface or not? This study propose to establish the unique turbulent flow shear stress environment of bladder urine based on our successful designs of bionic bacterial biofilm reactor model of human bladder and establish reliable mechanobiological pathogenesis model of bacterial biofilm associated infection. The negative biological response of disturbed flow shear stress derived from bladder urine to Escherichia coli would be investigated from the aspects of structure and function. The laser scanning confocal microscopy, scanning electron microscope, lectin microarray and gene chip technology would be used. The research contents include the growth curve of planktonic and biofilm bacteria, structure of bacterial biofilm on carrier surface, bacterial lectin expression profiling and biofilm bacteria gene expression profiling, so as to explore the role of bladder urine turbulent flow shear stress in this kind of infection, and provide ideas for further development of new anti-infection technology.

尽管已认识到细菌生物膜形成为生物医用材料感染的关键，但其形成机理仍未明确。活的生物体内所有器官、组织及细胞都在应力刺激的作用下处于一种生理应变状态，产生复杂的生物学效应，同时，力学过程对生物体损伤或疾病产生重要影响。课题组前期研究已证实静水压刺激大肠埃希菌在载体表面形成细菌生物膜，并呈时-量关系。那么，膀胱尿流动态力学刺激是否调控体内导管表面细菌生物膜形成？基于此，本研究拟以前期自行成功设计的仿人体膀胱细菌生物膜反应器构建膀胱独特的尿液紊流切应力环境，建立细菌生物膜力学生物学发病机制模型，联合应用激光共聚焦与电子显微镜、凝集素芯片及基因芯片技术，从结构、功能两方面研究该力学刺激大肠埃希菌的负向生物学响应，包括细菌生长曲线、载体表面细菌生物膜结构、细菌凝集素及生物膜细菌基因表达谱，探讨膀胱尿液紊流切应力在细菌生物膜感染中的作用，并望为进一步开发抗感染新技术提供思路。

尽管已认识到细菌生物膜形成为生物医用材料感染的关键，但其形成机理仍未明确。活的生物体内所有器官、组织及细胞都在应力刺激的作用下处于一种生理应变状态，产生复杂的生物学效应，同时，力学过程对生物体损伤或疾病产生重要影响。课题组前期研究已证实静水压刺激大肠埃希菌在载体表面形成细菌生物膜，并呈时-量关系。那么，膀胱尿流动态力学刺激是否调控体内导管表面细菌生物膜形成？基于此，本研究首次提出人体膀胱尿液为独特的紊流切应力概念，并以前期工作基础成功构建体外仿人体膀胱模型细菌生物膜反应器；通过蠕动泵调控尿速、尿液出入反应器调控尿量、电动搅拌机旋转搅拌调控尿流，三个维度联合模拟构建紊流切应力，设置静态与动态、生理与病理两类、四种流体应力，建立了完整的力学参数体系。联合应用激光共聚焦与电子显微镜、凝集素芯片及基因芯片技术，从结构、功能两方面研究该力学刺激大肠埃希菌的负向生物学响应。结构分析提示，通过大肠埃希模式菌生物膜细菌菌落计数、OD 值测定、CLSM 联合SEM 细菌生物膜表征，提示随着仿人体膀胱尿流不同应力作用时间延长及应力施加作用增加，菌膜生成显著增加，组间、组内差异明显；功能分析提示，菌膜主要结合到四种凝集素即AAL，RCA-I，HPA和LEL，糖基化结合信号强弱随着时间变化明显转化，主要表现在岩藻糖和末端N-乙酰半乳糖胺；仿人体膀胱尿流应力均较静水压及恒流应力刺激大肠杆菌生物膜差异基因表达明显，而差异表达基因主要富集于碳代谢、核糖体、精氨酸和脯氨酸代谢、2-氧羰基水杨酸代谢及氨基酸生物合成五种通路。本研究表明人体膀胱尿流紊流切应力在细菌生物膜感染中可能发挥较为重要的作用，为进一步开发抗感染新技术提供思路。