摘要
目的:探讨阿司匹林提高幽门螺杆菌(H pylori)对克拉霉素敏感性的机制.方法:二氧化硅法提取细菌基因组DNA,限制性片段长度多态性(PCR-RFLP)检测阿司匹林处理前后23SrRNA突变位点的变化.液体闪烁计数法与SDS-PAGE电泳法分别测定阿司匹林处理前后H pylori胞内[7-3H]四环素的放射活性和外膜蛋白的变化.实时定量PCR(Taqman探针法)测定编码H pylori孔蛋白基因(hopA、hopB、hopC、hopD和hopE)及外流泵蛋白基因(hefA、hefB和hefC)的变化.结果:阿司匹林不改变H pylori克拉霉素耐药菌株23SrRNA基因的2143A-G突变位点,其增加H pylori胞内放射活性.阿司匹林处理后H pylori外膜蛋白在分子量55-72kDa之间有两条带表达改变,其不改变H pylori孔蛋白基因hopA、hopB、hopC、hopD、hopE及外流泵蛋白基因hefA、hefB、hefC的mRNA水平.结论:阿司匹林可能通过改变H pylori外膜蛋白的表达,提高H pylori外膜对抗生素的通透性,增加H pylori胞体内抗生素浓度.
AIM： To investigate the mechanisms of aspirin increasing the susceptibility of H pylori to clarithromycin. METHODS： Mutations in V function domain of 23SrRNA gene were identified by polymerase chain reaction （PCR） and restriction fragment length polymorphism analysis. H pylori strain 26695 treated with or without aspirin was incubated with [7-^3H] tetracycline. Endocellular radioactivity at different time points was analyzed in a liquid scintillation counter. Outer membrane proteins （OMPs） of H pylori 26695 were depurated and analyzed by SDS-PAGE. Total RNA of H pylori 26695 was extracted and the cDNA of the 5 porins （hopA, hopB, hopC, hopD, hopE） and the putative RND efflux system （hefABC） were obtained by reverse tran-scription （RT）. The expression of the above 8 cDNAs were analyzed using Taqman-based real-time PCR. RESULTS： Position 2143A-G mutations in V function domain of 23SrRNA gene did not change in clarithromycin resistant strains treated with aspirin. The radioactivities of H pylori cells increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori to antimicrobials. The OMP prof iles of H pylori treated with aspirin were similar to that of controls. However, the expression of two OMPs between 55-72 kDa altered in the presence of aspirin. Irrespective of the presence of aspirin, the expression of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC did not change at the mRNA level. CONCLUSION： Aspirin may enhance the permeability of the outer membrane of H pylori to antimicrobials, and thus increase the endocellular concentrations of antimicrobials probably by altering the OMP expression.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第18期1990-1996,共7页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30470777~~
