慢性化脓性中耳炎大鼠中耳黏膜的细菌生物膜形成特点及意义

Formation characteristics and significance of bacteria biofilm in middle ear mucosa of rats with chronic suppurative otitis

目的:建立慢性化脓性中耳炎大鼠模型,观察细菌生物膜在模型动物中耳黏膜的形成特点,探讨其在慢性化脓性中耳炎发病机制中的作用。方法:将28只大鼠分为6个实验组(分别为接种菌液后1、3、6、10、15、21d组)和1个对照组,每组4只。所有实验组大鼠用浓度为1×106 cfu/ml的铜绿假单胞菌菌液经鼓膜穿刺途径感染双侧中耳腔。分别在接种细菌后1、3、6、10、15、21d,将相应实验组的4只大鼠麻醉,用电耳镜观察双侧鼓膜,对炎症的严重程度进行评分;大鼠处死后取双侧听泡,左侧听泡制备成扫描电镜标本并观察中耳黏膜细菌生物膜形态,右侧听泡采用激光共聚焦显微镜观察中耳黏膜细菌生物膜形态。对照组在第0天即处死取材。结果:①用扫描电镜和激光共聚焦显微镜观察大鼠中耳黏膜标本,均在接种细菌6d左右观察到符合各自判定标准的细菌生物膜形成,到10d后形态更加典型,并在3周内形态保持稳定;②用电耳镜观察大鼠鼓膜,发现炎症感染程度在接种菌液10d内逐渐加重,10d后达到高峰并持续至第3周。1d组、10d组与6d组鼓膜评分比较,差异有统计学意义(P<0.05、P<0.01)。结论:①接种菌液后1～5d是细菌黏附、聚集阶段,6d后细菌生物膜三维结构初步形成,10d左右达到成熟稳定状态;②随着中耳黏膜细菌生物膜的生长与成熟,中耳炎炎症也在逐渐加重,提示中耳炎炎症感染的严重程度与细菌生物膜的成熟程度密切相关。

Objective：To establish the rat model of chronic suppurative otitis media and observe the formation of bacterial biofilm in middle ear mucosa of animal models and discuss the role of bacterial biofilm in the pathogenesis of chronic suppurative otitis media.Method：Twenty-eight rats were divided into six experimental groups and a control group evenly.All rats in experimental groups were infected with pseudomonas aeruginosa solution in 1×106 cfu/ml concentration through the tympanic membrane puncture approach to bilateral middle ear cavity.On the first,sixth,tenth,fifteenth and twenty-first st day after inoculation respectively,four rats in one experimental group were narcotized,then two-sided tympanic membrane of each rat were observed by using electric otoscope.We rated on the severity of the inflammation from the general pathology level（0 for normal,four for the most se-rious）.After the execution,the two-sided otocysts were obtained.The left was made to SEM specimen and the shape of bacterial biofilm in middle ear mucous was observed.The right was observed by CLSM.Control group were executed at the beginning of the experiment.Result：①Bacterial biofilm in line with their respective criteria were found at the six days after the inoculation,and were more typical in shape after ten days.Then the states maintained stably within three weeks.②By observing tympanic membrane under electric otoscope,it can be seen that the inflammation severity of otitis media aggravated gradually in the first ten days and achieved the peak,then the state continued to the third week.The differences of tympanic membrane rating between one day group and six day group,six day group and ten day group were statistically significant（P0.05,P0.01）.Conclusion：①In this experiment,the process of bacterial biofilm development in rats model is： from beginning to the five days,the bacteria adhere and accumulates.After six days,the 3D structure of bacterial biofilm preliminary formatted.After ten days,the bacterial biofilm achieves the mature and steady state.②With the growth and maturity of bacterial biofilm in middle ear mucosa,the inflammation of otitis media is gradually increasing,which suggests that the inflammation severity of otitis media and the maturation of bacterial biofilm in middle ear mucosa are closely related.