负压创面疗法治疗铜绿假单胞菌感染创面的相关机制研究
发布时间：2018-08-30 15:05
【摘要】：研究背景与目的在医疗领域,皮肤软组织感染仍然是亟需解决的难题。由感染导致的创面延迟愈合、不愈合,病情加重,住院时间延长等等给患者带来了沉重的心理及经济负担。临床上,负压创面疗法被广泛用于处理开放伤可能伴有感染的患者,且方法有效。然而,原因与机制并不完全清楚。有研究发现负压环境下创面细菌数量会发生变化,这可能是负压创面疗法减轻感染的机制之一。而负压环境下铜绿假单胞菌的细菌数量、毒素毒力等变化机制目前相关研究甚少。本研究采用铜绿假单胞菌感染软组织损伤的模型,研究负压环境对铜绿假单胞菌体外体内增殖、毒素分泌、毒力表达以及生物膜的相关作用及其机制。研究方法1.创建铜绿假单胞菌PAO1体外增殖模型,观察负压对细菌增殖的相关作用。应用铜绿假单胞菌PAO1侵染软组织损伤动物模型,通过负压创面疗法与常规纱布治疗的对照研究,采用多点取材培养法分析创面软组织内细菌数量的变化趋势及其原因。2.采用上述动物模型,通过酶联免疫吸附法、浓硫酸-苔黑酚法、弹性蛋白-刚果红法检测创面软组织内毒素含量,通过扫描电镜、刀豆蛋白A染色等方法观察创面生物膜的变化。3.采用上述动物模型,通过实时荧光定量PCR (Real-time PCR)法评估铜绿假单胞菌毒素及生物膜调控基因表达变化。结果1.负压作用下铜绿假单胞菌在LB固体培养基上增殖24小时、48小时细菌生长直径均小于非负压组,p0.01。2.与常规纱布组相比,负压创面疗法治疗下创面软组织内细菌数量减少明显,在治疗的第四、六、八天软组织内细菌数量较常规纱布治疗组低,p0.01。但是两种治疗方法均没能将创面细菌数量降低到临床感染标准(10^5CFU/g组织)以下。3.与常规纱布组相比,负压创面疗法组创面软组织内细菌外毒素A、鼠李糖脂、弹性蛋白酶等毒素含量相对较低。4.负压创面疗法能够减少创面铜绿假单胞菌生物膜。5.负压创面疗法可以显著抑制铜绿假单胞菌toxA, rhlA, LasB, Lasl, Rh lI等基因表达水平。结论1.负压环境可以有效的抑制铜绿假单胞菌的增殖。负压创面疗法在一定程度上可以减少创面软组织内细菌数量,且效果优于常规纱布治疗。2.负压与引流的综合作用可以减少创面软组织内铜绿假单胞菌毒素的含量,且效果优于常规纱布治疗。负压环境可以抑制创面上铜绿假单胞菌毒素调控基因、生物膜调控基因的表达水平。3.负压创面疗法减轻铜绿假单胞菌创面感染的机制有抑制创面细菌增殖,抽吸引流创面细菌、毒素,抑制铜绿假单胞菌毒素及生物膜调控基因表达。
[Abstract]:Background and objective in medical field, skin and soft tissue infection is still a difficult problem to be solved. The delayed healing, nonunion, aggravation and prolonged hospitalization caused by infection bring heavy psychological and economic burden to the patients. In clinic, negative pressure wound therapy is widely used to treat open wound patients who may be associated with infection, and the method is effective. However, the reasons and mechanisms are not entirely clear. Some studies have found that the number of bacteria changes in the wound under negative pressure, which may be one of the mechanisms of negative pressure wound therapy to reduce infection. However, there are few studies on the change mechanism of Pseudomonas aeruginosa under negative pressure, such as the number of bacteria and toxin virulence. In this study, a model of soft tissue injury induced by Pseudomonas aeruginosa was used to study the effects of negative pressure on the proliferation, toxin secretion, virulence expression and biofilm of Pseudomonas aeruginosa in vitro. Method 1. To investigate the effect of negative pressure on the proliferation of Pseudomonas aeruginosa PAO1 in vitro. The animal model of soft tissue injury infected by Pseudomonas aeruginosa (PAO1) was used to analyze the change trend and cause of bacterial number in soft tissue of wound surface by using the method of multi-site culture through the comparative study of negative pressure wound therapy and routine gauze therapy. The endotoxin content in soft tissue of the wound was detected by Elisa, concentrated sulfuric acid / moss method, elastin / Congo red method, and scanning electron microscope (SEM). The changes of wound biofilm were observed by concanavalin A staining. The changes of gene expression of Pseudomonas aeruginosa toxin and biofilm were evaluated by real-time quantitative PCR (Real-time PCR). Result 1. Under negative pressure, the growth diameter of Pseudomonas aeruginosa on LB solid medium for 24 hours and 48 hours was smaller than that of non-negative pressure group (P 0.01.2). Compared with the conventional gauze group, the number of bacteria in the soft tissue of the wound treated with negative pressure wound therapy was significantly reduced, and the number of bacteria in the soft tissue on the 4th, 6th and 8th day of treatment was lower than that in the conventional gauze group. However, both treatments failed to reduce the number of bacteria in the wound to below the clinical infection standard (10 ^ 5CFU/g tissue). Compared with the conventional gauze group, the contents of bacterial exotoxin A, rhamnolipid and elastase in the wound soft tissue in the negative pressure wound therapy group were relatively lower than those in the negative pressure wound therapy group. Negative pressure wound therapy can reduce Pseudomonas aeruginosa biofilm. Negative pressure wound therapy could significantly inhibit the expression of toxA, rhlA, LasB, Lasl, Rh lI and other genes in Pseudomonas aeruginosa. Conclusion 1. Negative pressure environment can effectively inhibit the proliferation of Pseudomonas aeruginosa. Negative pressure wound therapy can reduce the number of bacteria in wound soft tissue to a certain extent, and the effect is better than that of routine gauze treatment. 2. The combined effect of negative pressure and drainage can reduce the content of Pseudomonas aeruginosa toxin in the soft tissue of wound, and the effect is better than that of routine gauze therapy. Negative pressure environment could inhibit the expression level of Pseudomonas aeruginosa toxin regulatory gene and biofilm regulatory gene. The mechanism of negative pressure wound therapy to alleviate the infection of Pseudomonas aeruginosa is to inhibit the proliferation of bacteria, draw and drain the bacteria and toxin, inhibit the expression of Pseudomonas aeruginosa toxin and biofilm regulatory gene.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R641
本文编号：2213438
[Abstract]:Background and objective in medical field, skin and soft tissue infection is still a difficult problem to be solved. The delayed healing, nonunion, aggravation and prolonged hospitalization caused by infection bring heavy psychological and economic burden to the patients. In clinic, negative pressure wound therapy is widely used to treat open wound patients who may be associated with infection, and the method is effective. However, the reasons and mechanisms are not entirely clear. Some studies have found that the number of bacteria changes in the wound under negative pressure, which may be one of the mechanisms of negative pressure wound therapy to reduce infection. However, there are few studies on the change mechanism of Pseudomonas aeruginosa under negative pressure, such as the number of bacteria and toxin virulence. In this study, a model of soft tissue injury induced by Pseudomonas aeruginosa was used to study the effects of negative pressure on the proliferation, toxin secretion, virulence expression and biofilm of Pseudomonas aeruginosa in vitro. Method 1. To investigate the effect of negative pressure on the proliferation of Pseudomonas aeruginosa PAO1 in vitro. The animal model of soft tissue injury infected by Pseudomonas aeruginosa (PAO1) was used to analyze the change trend and cause of bacterial number in soft tissue of wound surface by using the method of multi-site culture through the comparative study of negative pressure wound therapy and routine gauze therapy. The endotoxin content in soft tissue of the wound was detected by Elisa, concentrated sulfuric acid / moss method, elastin / Congo red method, and scanning electron microscope (SEM). The changes of wound biofilm were observed by concanavalin A staining. The changes of gene expression of Pseudomonas aeruginosa toxin and biofilm were evaluated by real-time quantitative PCR (Real-time PCR). Result 1. Under negative pressure, the growth diameter of Pseudomonas aeruginosa on LB solid medium for 24 hours and 48 hours was smaller than that of non-negative pressure group (P 0.01.2). Compared with the conventional gauze group, the number of bacteria in the soft tissue of the wound treated with negative pressure wound therapy was significantly reduced, and the number of bacteria in the soft tissue on the 4th, 6th and 8th day of treatment was lower than that in the conventional gauze group. However, both treatments failed to reduce the number of bacteria in the wound to below the clinical infection standard (10 ^ 5CFU/g tissue). Compared with the conventional gauze group, the contents of bacterial exotoxin A, rhamnolipid and elastase in the wound soft tissue in the negative pressure wound therapy group were relatively lower than those in the negative pressure wound therapy group. Negative pressure wound therapy can reduce Pseudomonas aeruginosa biofilm. Negative pressure wound therapy could significantly inhibit the expression of toxA, rhlA, LasB, Lasl, Rh lI and other genes in Pseudomonas aeruginosa. Conclusion 1. Negative pressure environment can effectively inhibit the proliferation of Pseudomonas aeruginosa. Negative pressure wound therapy can reduce the number of bacteria in wound soft tissue to a certain extent, and the effect is better than that of routine gauze treatment. 2. The combined effect of negative pressure and drainage can reduce the content of Pseudomonas aeruginosa toxin in the soft tissue of wound, and the effect is better than that of routine gauze therapy. Negative pressure environment could inhibit the expression level of Pseudomonas aeruginosa toxin regulatory gene and biofilm regulatory gene. The mechanism of negative pressure wound therapy to alleviate the infection of Pseudomonas aeruginosa is to inhibit the proliferation of bacteria, draw and drain the bacteria and toxin, inhibit the expression of Pseudomonas aeruginosa toxin and biofilm regulatory gene.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R641
【共引文献】
相关期刊论文 前1条
1 潘婷婷;李镛;刘嘉琳;瞿洪平;;铜绿假单胞菌感应系统基因表达与毒力因子的相关性分析[J];内科理论与实践;2015年04期
相关硕士学位论文 前1条
1 张美光;负压封闭引流联合含氧液冲洗治疗慢性创面的临床研究[D];南方医科大学;2014年
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