超声靶向微泡破坏增强人-防御素3抑制小鼠体内耐药葡萄球菌生物膜形成  

Enhancement of human-defensin 3 activity against antibiotic- resistant staphylococcus biofilms in mice model by ultrasound- targeted microbubble destruction

作　　者：朱晨[1] 方诗元[1] 孔荣[1] 禹德万[1] 黄炎[1] 夏睿[1] 徐玮[1] 王英明[1] 马锐祥[1] 刘应生[1] 李雷[1] 李多玉[1] 

机构地区：[1]安徽医科大学附属省立医院骨一科,安徽合肥230001

出　　处：《生物骨科材料与临床研究》2015年第5期8-11,15,I0004,I0005,共7页Orthopaedic Biomechanics Materials and Clinical Study

基　　金：国家自然科学基金(No:81401815;81301571;81271594)

摘　　要：目的探讨内源性抗菌肽人-防御素-3(HBD-3)联合低频超声(US)及造影剂微泡(MB)的靶向破坏(UTMD)在体内抑制耐药葡萄球菌生物膜形成的作用。方法钛片与2种耐药葡萄球菌共培养24小时待生物膜形成后,放置于小鼠后背部脊柱皮下的两侧;再通过液体微量稀释法,获取HBD-3对2种耐药菌的最小抑菌浓度(MIC)。48只小鼠按不同的处理条件随机分为6组:(a)0 g/m LHBD-3;(b)超声处理组;(c)微泡+超声处理组;(d)10 MIC HBD-3;(e)10 MIC HBD-3+超声处理组;(f)10 MIC HBD-3+超声+微泡处理组。术后3天,处死各组小鼠取出钛片。涂板计数、激光共聚焦显微镜及电子显微镜观察不同实验处理条件下,钛片生物膜的厚度及膜内的剩余活菌量。Realtime PCR定量分析相关成膜基因及耐药基因,并进行统计学分析。结果与其它治疗组相比,体内最高浓度的HBD-3联合UTMD组的活细菌数百分比明显下降。同时,超声微泡还能显著提高HBD-3抑制成膜相关基因ica AD以及耐甲氧西林基因Mec A的表达并同时促进ica R的表达。结论 UTMD可能有很大的潜力,提高HBD-3的抗菌活性并抑制小鼠体内的生物膜感染。Objectives To investigate the effect of Human β-defensin-3（HBD-3）, an endogenous antimicrobial peptide,combined with ultrasound（US）-targeted microbubble（MB）destruction（UTMD） on antibiotic-resistant Staphylococcus biofilms in vivo. Methods Two biofilm-infected titanium plates were implanted subcutaneously bilateral to the spine.Biofilms of the 2 antibiotic-resistant Staphylococcus strains from the plates in mice were treated under 6 different conditions：（a） untreated control,（b） US only,（c） US＋MB,（d） 10 MIC HBD-3,（e） 10 MIC HBD-3＋US and（f） 10 MIC HBD-3＋US＋MB. At 3 days after the surgery, the six treatments were completed, the mice were euthanized and the implants were taken out. The amount of bacteria in the biofilms were observed by the spread plate method, confocal laser scanning microscopy and scanning electron microscopes under different experiment conditions. Biofilm-associated and drug-resistant genes were quantitatively analyzed by real-time polymerase chain reaction, then statistical analysis was carried out. Results In the study, we demonstrated that the percentage of live cells, and the viable counts of 2 tested Staphylococcus recovered from the biofilms on the titanium surface in vivo were significantly decreased in the highest concentrations of HBD-3 combined with UTMD compared with those of any other groups. Moremore, we found that UTMD could enhance HBD-3 activity inhibiting the biofilm-associated genes expression of ica AD and the methicillin-resistance genes expression of Mec A by simultaneously promoting the genes expression of ica R. Conclusion UTMD may have great potential for improving HBD-3 antibiotic activity against Staphylococcus biofilms infections in vivo.

关 键 词：超声 微泡 葡萄球菌 生物膜 Β-防御素 

分 类 号：R96[医药卫生—药理学]