【摘要】 目的研究盐酸小檗碱对表皮葡萄球菌形成生物被膜所需基因的抑制作用和生物被膜形成前后蛋白质分子的表达差异。方法构建表皮葡萄球菌生物被膜阳性模型,用快速银染法鉴定盐酸小檗碱作用前后表皮葡萄球菌生物被膜形成情况;以1 000μg/mL盐酸小檗碱、3 mg/mL万古霉素、环丙沙星和红霉素分别处理生物被膜模型,采用real-time PCR方法检测药物作用前后表皮葡萄球菌icaA基因和agr基因的表达变化;用改良双向电泳技术对500μg/mL盐酸小檗碱作用前后细菌的总蛋白进行分离。结果在合适的培养条件下,表皮葡萄球菌生物被膜形成阳性,表皮葡萄球菌生物被膜模型构建成功。盐酸小檗碱对icaA基因和agr基因表达存在抑制作用,与未经药物处理组比较,P均<0.05;其中对agr基因的抑制作用不及万古霉素、环丙沙星及红霉素(P均<0.05),各药物处理组对icaA基因抑制作用无统计学意义。通过电泳分离,共得到23个差异表达的蛋白质分子,其中13个仅在药物处理组表达升高,有10个仅在非药物处理对照组表达升高。结论中药单体盐酸小檗碱对表皮葡萄球菌形成生物被膜关键基因的表达具有弱的抑制作用,通过二维电泳分离得到的差异表达蛋白分子为后续细菌耐药机制的研究奠定基础。更多还原

【Abstract】 Objectives To explore the inhibitive effects of berberine hydrochloride on the related gene expression during the biofilm forming of Staphylococcus epidermidis and the differentiated expression of proteins before and after the formation of biofilm.Methods The biofilm forming model of Staphylococcus epidermidis was constructed and identified by rapid argent nitrate staining.Following the treatments of the model with 1 000 μg/mL berberine hydrochloride,500 μg/mL vancomycin,ciprofloxacin or erythromycin respectively,the expressions of icaA and agr gene were analyzed by real-time PCR.Before and after the treatment of Staphylococcus epidermidis with 500 μg/mL berberine hydrochloride,the total protein of the bacteria was extracted and separated by modified two-dimensional electrophoresis(2-DE),and the protein points of differentiated expression were identified by HPLC-CHIP/MS method.Results Under the appropriate cultural conditions,the biofilm of Staphylococcus epidermidis was formed,indicating the model was established successfully.After the model was treated with berberine hydrochloride,the expressions of icaA and agr gene were inhibited(P<0.05),while treatment of vancomycin,ciprofloxacin or erythromycin induced stronger inhibitive effects for agr gene in the model than berberine hydrochloride(P<0.05).Similar inhibitive effects for icaA gene were shown by berberine hydrochloride and other 3 antibiotics(P>0.05).Modified 2-DE analysis showed 23 differentially expressed proteins,which were identified further by HPLC-CHIP/MS.Among these differentiated proteins,13 were up-regulated expression after treatment of berberine hydrochloride.Conclusions There was a weak inhibition of berberine hydrochloride on the key genes for biofilm forming of Staphylococcus epidermidis.The identification of differentially expressed proteins provided experimental data for the further exploration on the mechanism of bacterial antibiotic resistance.更多还原