RNA干扰铜绿假单胞菌群体感应系统对生物膜影响的研究

The effect of small interfering RNA against quorum sensing system of Pseudomonas aeruginosa on the biofilm

目的观察小干扰RNA(small interfering RNA,siRNA)分子干扰铜绿假单胞菌群体感应系统后,对铜绿假单胞菌生物膜的影响。方法首先针对lasR、rhlR和rhlI基因设计4条siRNA分子,分别是si-asr1、si-hlr1、si-hlr2和si-hli1,体外转录法制备上述siRNA分子。将50nmol/LsiRNA分子与4μl脂质体混合,然后转染铜绿假单胞菌成熟期生物膜,应用平板菌落计数法检测siRNA转染后96h内生物膜活细菌数目;siRNA转染生物膜48h后,应用1%复红染色法光学显微镜观察生物膜形态改变,同时应用RT-PCR法检测生物膜弹性蛋白酶B基因lasB表达量。结果si-hlr1、si-hlr2和si-hli1分别转染铜绿假单胞菌生物膜48h,生物膜出现解体,生物膜内活细菌数目无明显改变。si-asr1转染铜绿假单胞菌生物膜48h后,生物膜形态及生物膜内活细菌数目无明显改变。si-asr1、si-hlr2和si-hli1分别转染铜绿假单胞菌生物膜48h,生物膜lasB基因表达量分别下调了49%、21%和18%。si-hlr1转染生物膜48h后,lasB基因表达量无明显改变。结论应用siRNA分子干扰铜绿假单胞菌群体感应系统的lasR、rhlR和rhlI三个关键性基因,可以促进生物膜解体,同时能部分抑制生物膜lasB基因表达,但是不能减少生物膜内活细菌数。

Objective To investigate the effect on biofilm by small interfering RNA （siRNA） against quorum sensing system of Pseudomonas aeruginosa. Methods Four siRNA（si-asr1, si-hlr1, si-hlr2 and si-hli1） against lasR, rhlR and rhll genes, were designed and prepared by transcription in vitro. After the mature biofilm of Pseudomonas aeruginosa were transfected with the mixture of 50 nmol/L siRNA and 4μ1 lipofect, the number of living bacteria in biofilm was calculated by plate culture method during 96 hours. 48 hours after transfection, the morphology changes of biofilm were observed by light microscope after 1% azaleine staining, and the expression level of elastase B gene （lasB） was determined by RT-PCR. Results 48h after the biofilm of Pseudomonas aeruginosa alone transfected with sihlrl, si-hlr2 and si-hli1, the biofilm disassembly emerged, and the number of living bacteria in biofilm didn＇t change obviously. 48h after the biofilm of Pseudomonas aeruginosa alone transfected with si-asr1, the morphology of biofilm and the number of living bacteria in biofilm didn＇t change obviously. 48h after the biofilm were alone transfected with si-asr1, si-hlr2 and si-hli1, the expression level of lasB were decreased by 49%, 21% and 18%, respectively. 48h after the biofilm alone transfected with si-hlr1, the expression level of lasB didn＇t change obviously. Conclusions siRNA against quorum sensing system of Pseudomonas aeruginosa enhanced the disassembly of biofihn, and partly inhibited the expression of lasB gene in biofilm, but didn＇t reduce the number of living bacteria in biofilm.