siHybrids技术沉默铜绿假单胞菌外排泵mexB基因体外效应的初步研究  

A preliminary study on siHybrids technique on inhibiting the effiux pump gene mexB of Pseudomonas aeruginosa in vitro

作　　者：毛艳[1] 陈佳[1] 许东[1] 邢铭友[1] 王丽丽[1] 谢旭华[1] 龚凤云[1] 夏超[1] 申爱霞[1] 宋莹[1] 占伟丽[1] 宋建新[1] 

机构地区：[1]华中科技大学附属同济医院,武汉430030

出　　处：《中华微生物学和免疫学杂志》2011年第8期707-712,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30873189）

摘　　要：目的初步研究siHybrids技术对铜绿假单胞菌野生菌PA01外排泵mexB基因体外沉默效应。方法针对铜绿假单胞菌野生株PAOI外排泵mexB基因设计并合成3条特异性silly—brids分子和l条阴性对照siHybrids分子。在分子浓度为50nmol／L下，分别以合成的siHybrids分子干扰铜绿假单胞菌野生菌PA01，并设实验组为铜绿假单胞菌野生菌PA01空白对照组，阴性对照组scamble（SC）-001，干预组siHybrids（si）-001、siHybrids（si）-002及siHybrids（si）-003，分别在干预12h及24h后采用real．timePCR法检测各实验组中靶基因mexB基因mRNA的表达水平。进一步采用Mueller-Hinton倍比稀释法检测50nmol／L浓度下，siHybrids分子干预铜绿假单胞菌野生菌PA01前后氯霉素（CP）、红霉素（EM）、左氧氟沙星（L-OFLX）、头孢他啶（CAZ）、美洛培南（MER）的最小抑菌浓度（Mm）值。结果不同siHybrids分子干预PA0112h后，mexB基因mRNA表达量无明显差异性；但干预24h后，mexB基因mRNA表达量：干预组（si-001，si-002，si-003）比空白对照组、阴性对照组（sc-001）有明显下降。对比干预12h、24h后mexB基因mRNA表达量，可以发现空白对照组、阴性对照组（sc-001）mRNA的表达量成上升趋势，而干预组（si-001，si-002，si-003）mexB基因mRNA表达量均呈下降趋势。siHybrids分子在干预铜绿假单胞菌野生菌24h前后的氯霉素（CP）、红霉素（EM）、左氧氟沙星（L—OFLX）、头孢他啶（CAZ）、美洛培南（MER）MIC无明显差异性。结论在mRNA表达水平上，siHybrids分子能体外干预铜绿假单胞菌PA01mexB基因mRNA表达，此种沉默作用呈现时间依赖性，且在24h能有效地发挥干预作用。Objective To investigate the effect and mechanism of siHybrids technique on inhibi- ting the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro. Methods Targeting the efflux pump gene mexB of Pseudomonas aeruginosa PAO1, we designed and synthesized three siHybrids molecule and one negative scanle siHybrids molecule. Pseudomonas aeruginosa PA01 were intervened by the siHy- brids molecules in 50 nmoL/L, respectively. And the experiments were made of control groups [ blank and scamble（sc） -001 ] and intervened groups[ siHybrids （si） -001, siHybrids （si） -002 and siHybrids （si） -003 ] of Pseudomonaa aeruginosa PAO1. The targeting efflux pump gene mexB mRNA expressions of Pseudomonas aeruginosa PAO1, including all groups, were measured by real-time PCR in 12 h and 24 h after interference in vitro. Further,the minimal inhibitory concentration（MIC） of chlormycetinCP, erythrocin（ EM）, levoflox- acin （L-OFLX）, ceftazidime （CAZ）, meropenem（MER） to those groups were detected by using Mueller-Hin- ton broth dilution before and after interference. Results The relative mexB mRNA amounts of Pseudomonas aeruginosa PAO1 intervened by different siHybrids were not much more different from each other after 12 h, but the expression of mexB mRNA of the intervened group （si-001 ,si-002, si-003 ）was much lower than control groups after interference for 24 h. The relative mexB mRNA amounts, comparing 12 h with 24 h, we would find the blank control and negative control submit escalating trend. And the intervened control, three different siHybrids were all with a downward tendency. However, in presence of 50 nmol/L siHybrids, the minimal inhibitory concentration（MIC） of CP, EM, L-OFLX, CAZ, MER to those controls were not much more different before and after interference. Conclusion On level of the mRNA expressions, siHybrids could inhibit the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro, and within 24 h would be able to function effectively. Further, the effect was tim

关 键 词：siHybrids技术 RNAI 铜绿假单胞菌 外排泵 REAL-TIME PCR 

分 类 号：R446.5[医药卫生—诊断学]