siHybrids分子对耐药肺炎克雷伯菌kpc-2抑制作用研究  

A study of si Hybrids on inhibiting the kpc-2 gene of Klebsiella pneumoniae

作　　者：范芳华[1] 吴国友[1] 周筱琼[1] 肖震[1] 程璐[1] 

机构地区：[1]浙江医院检验科细菌室

出　　处：《中国临床药理学与治疗学》2016年第5期490-494,共5页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：浙江省医药卫生科技计划项目(2015KYA010)

摘　　要：目的:本研究采用RNA干扰技术和合成小分子抑制剂的方法,研究si Hybrids分子对产碳青霉烯酶肺炎克雷伯杆菌耐药性的影响。方法:通过对临床分离的48株肺炎克雷伯杆菌进行药敏实验,初步筛选并收集了耐碳青霉烯类抗生素的菌株;进一步利用PCR检测,筛选出携带碳青霉烯酶基因kpc-2的耐药株。利用si Hybrids技术,针对kpc-2设计合成si Hybrids杂合分子,对肺炎克雷伯菌碳青霉烯酶基因kpc-2进行特异性沉默后,通过荧光定量PCR实验检测si Hybrids沉默靶基因kpc-2的体外表达水平,并通过联合抑菌实验验证si Hybrids分子提高抗生素抗菌活性乃至逆转细菌耐药性的效果。结果:合成的小分子siHybrids可以使耐药肺炎克雷伯杆菌kpc-2基因的表达下调,并且与碳青霉烯类抗生素亚胺培南、美罗培南、厄他培南分别联合使用,可使肺炎克雷伯杆菌对亚胺培南、美罗培南、厄他培南由耐药转为敏感。结论:si Hybrids可以特异地抑制耐药肺炎克雷伯杆菌kpc-2基因的表达,通过干扰产碳青霉烯酶基因kpc-2造成肺炎克雷伯杆菌对碳青霉烯类抗生素耐药性的逆转。AIM：This research used sythesized micromolecules siHybrids to study on their influence on kpc-2-mediated resistance in Klebsiella pneumoniae.METHODS：48 clinical isolated strains of Klebsiella pneumoniae were tested for drug sensitivity and the carbapanems resistant ones were chosen for selecting the kpc-2-carrying strains by polymerase chain reaction （PCR）,which were indentified as kpc-2 mediated carbapanems resistant ones.By the techonology of siHybrids,a siHybrids molecule was designed and sythesized and a scamble siHybrids molecule as the negative control.The relative transcription of kpc-2 gene in Klebsiella pneumoniae cultured with or without siHybrids were detected by quantitative real-time PCR,and the minimal inhibitory concentrations （MICs） of Imipenem,Meropenem,Ertapenem to Klebsiella pneumoniae were determined by using Mueller-Hinton broth dilution before and after siHybrids interference,respectively.RESULTS：The relative transcriptional level of kpc-2 in Klebsiella pneumoniae intervened by siHybrids was lower than that of untreated stain.Correspondingly,the antibiotic susceptibility revealed that the intervened strains are more sensitive to imipenem,meropenem and ertapenemwhen compared with the the original samples.CONCLUSION：The sythesized micromolecule siHybrids could inhibit the expression of kpc-2,which effectively inverted the antibiotic resistance of carbapanems resistant Klebsiella pneumoniae.

关 键 词：肺炎克雷伯杆菌 碳青霉烯酶 kpc-2 siHybrids分子 

分 类 号：R446.5[医药卫生—诊断学]