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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。
Objective To investigate the effects of endogenous antimicrobial peptide human defensin-3 (HBD-3) combined with low-frequency ultrasound (US) and targeted microbubble (MB) targeted disruption (UTMD) on the formation of drug-resistant staphylococcal biofilm effect. Methods Titanium plates were co-cultured with two kinds of drug-resistant Staphylococcus aureus for 24 hours to form biofilm and then placed on both sides of the posterior spine of the back of the mice. Then liquid micro-dilution method was used to obtain the biofilms of HBD- Minimum inhibitory concentration (MIC). Forty-eight mice were randomly divided into 6 groups according to different treatment conditions: (a) 0 g / m LHBD-3; (b) sonicated group; (c) microbubble + sonicated group; 3; (e) 10 MIC HBD-3 + ultrasound treatment group; and (f) 10 MIC HBD-3 + ultrasound + microbubble treatment group. Three days after operation, all mice were sacrificed and titanium tablets were removed. The thickness of the biofilm and the amount of remaining viable bacteria in the membrane were observed under different experimental conditions by using laser scanning confocal microscope and electron microscope. Realtime PCR quantitative analysis of genes related to film-forming and drug resistance, and statistical analysis. Results Compared with other treatment groups, the highest percentage of live bacteria in the body with HBD-3 in combination with UTMD significantly decreased the percentage of viable bacteria. At the same time, ultrasound microbubbles can significantly improve the expression of ica AD and methicillin-resistant gene Mec A and promote the expression of ica R. Conclusion UTMD may have great potential to improve the antimicrobial activity of HBD-3 and inhibit biofilm infection in mice.