摘要
目的探讨RNA分子干扰铜绿假单胞菌MexAB—OprM外排泵MexB基因的体内效应。方法针对MexAB-OprM外排泵MexB基因设计siRNA小分子，构建干扰RNA（shRNA）表达载体，将带有小分子干扰RNA（siRNA）质粒电转入铜绿假单胞菌，从小鼠支气管内直接予以siRNA质粒干扰PAO琼脂菌体悬液（1×10^7CFU／ml）攻击，建立PA01野生型及siRNA2干扰型和siRNA阴性对照分子干扰型菌株的肺部感染动物模型。于感染后第1、2、3天腹腔注射美罗培南（100mg／kg，2次／d）。感染后第3、5、7天评估各组小鼠肺部细菌学、肺部病理学、细胞因子水平及肺组织中性粒细胞募集（MPO）水平变化。结果siRNA2干扰组小鼠，感染后3、5、7d的肺部细菌负荷明显下降。且与siRNAnon组和ScrambledsiRNA组比较，siRNA2干扰组小鼠感染早期（3d）病理学改变明显减轻；MPO及炎性细胞因子（IL-1β和IL-12）表达水平升高。而在感染后期（7d）siRNA2干扰组小鼠炎性细胞因子表达水平则下降。结论siRNA分子干扰明显增加小鼠对铜绿假单胞菌感染的抵抗力，降低肺部细菌的载量，增加早期中性粒细胞募集到感染部位并诱导铜绿假单胞菌的早期免疫应答。siRNA分子联合抗生素的治疗，为慢性铜绿假单胞菌肺部感染的治疗提供了有意义的资料。
Objective To investigate the efficacy of small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM multidrag effiux pump in vivo. Methods Two short hairpin （sh） RNA expression vectors targeting the MexB gene, and negative controls, were designed, synthesized, and elec- trotransformed into the P. aeruginosa strain PAO1. The in vivo therapeutic efficacy of the MexB small interfer- ing （si）RNAs was determined by infecting a murine model of chronic P. aeruginosa lung infection（ lxl07 CFU/ml）. The mice were killed on day 3,5 and 7 after infection with the Pseudomonas aeruginosa str^ns. Results In the murine infection model, treatment with MexB-siRNAs led to significantly reduced bacteria burden of the bellows by day 5 and 7 post-infection, and reduced the P. aerug/nosa-induced pathological changes. In addition, MexB-siRNA2 treatment enhanced neutrophil recruitment and production of inflammato- ry eytokines （IL-1 β, IL-12） in the early infection stage （day 3 ） （P〈0.05）, both of which decreased by day 7. Conclusion MexB-siRNA could inhibit beth mRNA expression and the activity of P. aeruginosa in vitro. siRNA was effective in reducing the bacterial load in a murine model of chronic lung infection. Targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2012年第11期944-948,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（30873189）
