siRNA干扰铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达的研究  

The effect of small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM effiux pumps

作　　者：龚凤云[1] 王丽丽[1] 宋莹[1] 邢名友[1] 宋建新[1] 

机构地区：[1]华中科技大学同济医学院同济医院感染科,武汉430030

出　　处：《中华微生物学和免疫学杂志》2011年第9期796-800,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30873189）

摘　　要：目的探讨RNA干扰技术对铜绿假单胞菌MexA—MexB-OprM外排泵mexB基因表达及有关功能的影响。方法设计并合成4条小分子干扰RNA（small interfering RNA，siRNA）（siRNAl、siRNA2、siRNA3和siRNA4）序列，构建干扰RNA（short hair pin RNA，shRNA）表达载体，将带有siRNA质粒电转入PAOI和临床耐药株PA03。应用E-test方法检测PA01和PA03抗生素MIC的变化。用半定量RT—PCR和定量PCR方法检测mexB的表达变化。结果经酶切鉴定siRNA表达载体构建成功，并明显提高PAO和临床耐药株PA03对抗生素的敏感性。RT-PCR结果显示干扰后的PA01和临床耐药株PA03mexB基因表达明显下降（P〈0．05）。结论siRNA成功干扰MexA－MexB－OprM外排泉mexB基因的表达，提高铜绿假单胞菌对抗生素的敏感性，为临床上治疗铜绿假单胞菌感染，降低其耐药性提供了新的思路。Objective To identify the efficacy small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps. Methods Four siRNA （ siRNA1, siRNA2, siRNA3 and siRNA4） against mexB gene were designed and prepared by electricity transference in vitro. MICs of antibiotic combined with efflux pump inhibitors against multiple resistant strain PAO1 and PAO3 were determined by E-test method. The mRNA expression levels of efflux pump gene （mexB） were quantified by real time fluorescent quantitative PCR. Results siRNA expression vectors were constructed success by enzyme cut method. 48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA4, the sensibilities to anti- biotic were enhanced. 48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA1, siRNA2 and siRNA3, the sensibilities to antibiotic didn＇t change obviously. 48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA4, the expression level of mexB was decreased obviously （P 〈 0.05）. 48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA1, siRNA2 and siRNA3, the expression level of mexB didn＇t change obviously. Conclusion siRNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps enhanced the sensibility to antibiotic and inhibited the expression of mexB gene . Our results demonstrate the using RNAi may be potential largets for novel therapies directed against treatment of antibiotic-resistant infections.

关 键 词：siRNA分子 铜绿假单胞菌 MexA-MexB-OprM外排泵 

分 类 号：R516[医药卫生—内科学]