模拟人体膀胱体外构建仿生尿流应力细菌生物膜动态孵育系统

Simulating the human bladder to construct a bacterial biofilm dynamic incubation system under biomimetic urinary flow stress in vitro

摘要:

目的 模拟人体膀胱体外构建仿生尿流应力细菌生物膜动态孵育系统，探讨该系统的应用效果。方法 体外构建细菌生物膜动态孵育系统，采用大肠埃希菌为实验菌，硅胶膜片为成膜载体，建立5种尿流应力输出：生理白天和夜间分泌、生理白天排尿、病理白天和夜间排尿；设置2组仿生尿流应力流转方案：生理组和病理组。观察仿生尿流应力刺激下大肠埃希菌生物膜形成特点。检测方法包括：生物膜细菌活菌计数，荧光染色结合激光共聚焦显微镜观察，测定生物膜细菌荧光图像平均光密度值，构建生物膜立体图测量其厚度。测值多组间均数比较采用单因素方差分析（one-way analysis of variance，ANOVA），组内均数两两比较采用LSD-t法。结果 各组内比较结果显示，随孵育时间延长，生物膜细菌活菌计数值均逐渐增加，病理组48 h、静态组和生理组60 h显著增加并达到峰值（^cP=0.000）；生物膜细菌荧光图像平均光密度值和生物膜厚度测值均逐渐增加，病理组48 h、静态组和生理组72 h显著增加并达到峰值（^cP=0.000）；各组内其余测值差异无统计学意义（P>0.01）。各组间比较结果显示，与静态组比较，生理组和病理组36 h、48 h、60 h、84 h、96 h生物膜细菌活菌计数值均显著增加（^aP=0.000），与生理组比较，病理组增加更为显著（^bP=0.000）；与静态组比较，生理组96 h，病理组48 h、96 h生物膜细菌荧光图像平均光密度值显著增多（^aP=0.000），生理组72 h，病理组48 h、96 h BF厚度测值显著增多（^aP=0.000）；与生理组比较，病理组48 h、96 h细菌荧光图像平均光密度值和生物膜厚度测值均显著增多（^bP=0.000）；各组间其余测值差异无统计学意义（P>0.01）。结论 本研究模拟人体膀胱体外成功构建细菌生物膜动态孵育系统，经验证仿生尿流应力显著促进大肠埃希菌生物膜形成。

Abstract:

Objective To construct a dynamic incubation system of biomimetic urinary flow stress bacteria biofilm by simulating human bladder in vitro，and to explore the application effect of this system.Methods For the dynamic incubation system to construct bacterial biofilm in vitro，we used Escherichia coli as the experimental bacteria and silica gel membrane as the carrier of membrane formation to establish five bionic effects of urinary flow stress： physiological secretion during the day and night，physiological urination during the day， and pathological urination during the day and night. In addition， we set up two circulation scheme groups： physiological group and pathological group to observe the characteristics of biofilm formation of Escherichia coli under the stimulation of bionic urinary flow stress. The detection methods included： counting the living bacteria of biofilm bacteria， fluorescence staining combined with laser confocal microscope observation，determining the average light density value of the fluorescence image of biofilm bacteria，and constructing biofilm stereogram to measure the thickness of biofilm. The average values among multiple groups were detected by using one-way analysis of variance（ANOVA），and LSD-t method was used to compare the average values within groups in pairs.Results Through the comparison of data in each group，with the prolongation of incubation time，the number of viable bacteria of biofilm bacteria increased gradually，and the number of viable bacteria in pathological group at 48 h，static group and physiological group at 60 h increased significantly and reached the peak（^cP=0.000）；The measured values of average light density and biofilm thickness in the fluorescence image of biofilm bacteria increased gradually，and the measured values in the pathological group at 48 h，the static group and the physiological group at 72 h increased significantly and reached the peak（^cP=0.000）；There was no significant difference in other measured values in each group（P>0.01）. Through the comparison of data between groups，compared with the static group，the viable counts of biofilm bacteria in the physiological group and the pathological group at 36 h，48 h，60 h，84 h and 96 h were significantly increased（^aP=0.000）. Compared with the physiological group， the increase in the pathological group was more significant（^bP=0.000）. Compared with the static group， the fluorescence image average light density of biofilm bacteria in the physiological group at 96 h and the pathological group at 48 h and 96 h increased significantly（^aP=0.000），and the measured values of biofilm thickness in the physiological group at 72 h， pathological group at 48 h and 96 h increased significantly（^aP=0.000）. Compared with the physiological group， the measured values of the fluorescence image average light density and biofilm thickness of bacteria in the pathological group at 48 h and 96 h were significantly increased（^bP=0.000）. There was no significant difference in other measured values among the groups（P>0.01）.Conclusion In this study，a dynamic incubation system of bacterial biofilm has been successfully constructed in vitro to simulate human bladder， which has been verified that the biomimetic urinary flow stress can significantly promote the formation of Escherichia coli biofilm.