【作者】 柴栋；

【导师】 王睿；

【作者基本信息】 中国人民解放军军医进修学院 ， 药理学， 2002， 硕士

【摘要】 目的：本课题研究铜绿假单胞菌生物被膜相关感染的免疫逃逸现象，探讨免疫逃逸机理，并阐明克拉霉素以及加替沙星在防治生物被膜感染免疫逃逸中的作用。 方法：应用藻酸盐-粘液型铜绿假单胞菌接种免疫抑制SD大鼠，制备肺部细菌生物被膜感染病理模型；应用ELISA法测定血清抗铜绿假单胞菌抗体效价；应用菌落计数法测定肺部感染存活菌数；应用光镜、透射电镜观察生物被膜感染病理学改变；应用趋化试验、氧化爆发试验、淋巴细胞转化试验测定外周血中性粒细胞、淋巴细胞功能；应用结晶紫染色法、浓度梯度渗透法测定细菌粘附性、生物被膜渗透性；MTT法测定细菌生物被膜中存活菌数；应用葡萄糖醛酸内酯法测定细菌藻酸盐含量；应用改良组织培养平板法建立生物被膜体外模型；应用扫描电镜观察抗菌药物对生物被膜形态结构的影响。 结果：应用藻酸盐-粘液型铜绿假单胞菌珠接种免疫抑制SD大鼠，感染两周后，光镜下可见肺组织内淋巴细胞、中性粒细胞浸润，形成慢性炎性结节，透射电镜可见杆状细菌，周围藻酸盐包裹形成生物被膜。肺组织培养可见铜绿假单胞菌生长，大鼠外周血白细胞数目、比例正常，血清中有高效价抗铜绿假单胞菌抗体。5μg／ml铜绿假单胞菌藻酸盐可使中性粒细胞趋化作用减少10.57％、杀菌作用降低22.33％，并使淋巴细胞转化作用减弱25.70％，此抑制作用随藻酸盐浓度升高而增强。1／4MIC克拉霉素可使铜绿假单胞菌对硅胶片的粘附性降低15.7％，并提高加替沙星对生物被膜的渗透性33.6％～42.9％，与加替沙星联合应用可使生物被膜内存活菌数光密度值从0.281下降到0.224。10μg／ml克拉霉素可使铜绿假单胞菌藻 铜绿假单胞菌生物被膜感染免疫逃逸与防治 中文摘要 酸盐的分泌减少 12．4％。单独或联合应用克拉霉素＊ X加替沙星 o5m叭gy疗大鼠肺部细菌生物被膜感染＝周后，可使肺内存活菌数由 7．70opfu／g分别降至 5．81，3．80，0 lgCfu／g，抗体滴度（＞值）由 2．22 下降至l．56、l．41或l．08，肺部慢性炎症减轻，其中联合治疗组表现为慢 性炎症修复期改变。 结论：应用藻酸盐－粘液型铜绿假单胞菌接种免疫抑制SD大鼠，肺 部感染两周后，可建立大鼠肺部细菌生物被膜感染体内模型；铜绿假单胞 菌生物被膜感染过程中，存在免疫逃逸现象；此免疫逃逸现象与铜绿假单 胞菌藻酸盐密切相关；克拉霉素可抑制细菌藻酸盐的分泌；从而降低细菌 粘附性，提高加替沙星对生物被膜渗透性，并防止免疫逃逸现象产生。困 而克拉霉素与加替沙星联合治疗生物被膜相关感染具有协同作用。更多还原

【Abstract】 Obejective: To investigate the evidence evasion of host defense in Pseudomonas aeruginosa biofilm associated infection., and the mechanism of clarithromycin combined with gatifloxacin in therapy.Method: The animal model of biofilm associated chronic pulmonary infection was established by inoculating alginate - Pseudomonas aeruginosa to cortisone - treated rats. Biofilm model in vitro was established by the modified plate culture method. The antibody to Pseudomonas aeruginosa in sera was measured by ELISA method; The numbers of Pseudomonas aeruginosa in lung were determined by column counting method; Pathology changes of lung were observed by optic microscope and transmission electronic microscope (TEM) .The functions of neutrophil and lymphocyte were determined by locomotion test, chemiluminescence test and lymphocyte transformation test. The adherence of mucoid Pseudomonas aeruginosa was determined by crystal violet stain method; the gatifloxacin penetration activity against biofilm was determined by concentration gradient penetration method and the viable counts of bacteria in BF treated by gatifloxacin and clarithromycin were detected by MTT method. The quantity of alginate was calculated by standard curve of glucuronolactone method. The effect of antibiotics against biofilm conformation was observed by scan electronic microscope (SEM).Results: In the model of lung infections in cortisone-treated, inoculating alginate - Pseudomonas aeruginosa to cortisone - treated rats, lymphocyte and neutrophil infiltration, chronic granulomatous inflammation were observed in lung tissue by optic microscope, whereas bacilli surrounding by alginate and biofilm were found by TEM. The numbers and the ratio of peripheral blood leukocyte were normal and the antibody level to pseudomonas aeruginosa was high. In vitro we observed neutrophil locomotion, chemiluminescence and lymphocyte transformation were significantly reduced by Sug/ml Pseudomonas aeruginosa alginate by 10.57%,22.33% and 25.70%, 1/4MIC clarithromycin decrease the adhesion of Pseudomonas aeruginosa by 15.7% and increase penetration of gatifloxacin by 33.6%~42.9%. when gatifloxacin combined with 1/4MIC clarithromycin , the optical density of biofilm was decreased from 0.281 to 0.224.10 ug/ml clarithromycin inhibit the production of alginate at 12.4%. Compared with untreated group, treatment of clarithromycin or/and gatifloxacin decrease the viable counts of bacteria in lung from 7.70 Igcfu/g to 5.81, 3.80 , 0 Igcfu/g, and the antibody from 2.22 to 1.56,1.41 and 1.08. The pathology changes improved too, especially in combined treatment group.Conclusion: The animal model of biofilm associated chronic p M Imonary infection can be established by inoclating alginate - Pseudomonas aeruginosa to cortisone - treated rats. Evasion of host defense was correlated with Pseudomonas aeruginosa alginate. Clarithromycin inhibits the product of alginate, decreases the adhesion of Pseudomonas aeruginosa, increase the penetration of gatifloxacin through biofilm, and prevent biofilm evasion of host defense. So the combination of clarithromycin and levofloxacin issynergism in treating biofilm-associated chronic respiratory infection in an animal model. This may be a new strategy for the treatment of such infections in humans.更多还原