负压创面疗法治疗感染性软组织损伤相关机制研究

发布时间：2018-06-02 20:03

本文选题：感染 + 负压创面疗法　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：研究背景创面感染一直是骨科领域长期致力解决的难题。前期研究认为：感染是细菌与宿主体内环境相互作用的复杂过程；金葡菌致病力不仅与数量有关，更取决于细菌菌群状态和毒力因子表达。而且病原菌会适应环境变化调节相应的致病基因表达模式。负压创面疗法是一种能够有效治疗感染性软组织损伤的治疗方法，通过负压引流作用可在不减少细菌数量的情况下，显著控制感染，但其内在机制尚不明确。本研究拟采用金黄色葡萄球菌感染软组织损伤动物模型，通过研究负压创面疗法对金葡菌感染行为、群体感应agr系统、毒力状态以及机体固有免疫系统的作用机制，深化细菌性感染控制的认识与理论。目的 1.探讨负压创面疗法作用下细菌感染行为的改变与组织内即时细菌数量的变化趋势；2.探讨负压引流作用对金葡菌上游群体感应系统的影响，以及毒力因子表达与含量的变化；3.通过对炎性因子表达，固有免疫细胞趋化聚集的研究，探索负压创面疗法对机体固有免疫系统的影响。方法 1.建立金葡菌感染软组织开放性损伤动物模型,通过常规无菌纱布治疗与负压创面疗法的对照，应用扫描电镜（SEM）、透射电镜（TEM）、激光共聚焦显微镜（LSCM）等方法分析创面表面及组织内菌群形态、侵袭深度及数量变化，评估细菌感染行为的变化。 2.应用上述实验模型与方法，通过实时荧光定量PCR（Real-time PCR）、免疫印迹分析（Western Blot）评估金葡菌群体感应agr系统活性，并检测毒力因子的表达及含量。 3.检测创面组织内炎性因子IL-1β、IL-8和TNF-α的表达，对标本进行病理学检查，并定量分析不同组织深度内中性粒细胞聚集的数量。结果 1.相比无菌纱布治疗，，负压创面疗法作用下创面表面和组织内细菌均呈散在分布，且随治疗时间延长逐步明显；微菌群中的金葡菌散在分布，增生不活跃；同时细菌侵袭深度显著减低。 2.无菌纱布治疗与负压创面疗法两组在治疗day8时，创面细菌数量分别为治疗前的141.9±15.4%和34.6±5.5%(P＜0.01)；但根据相对比值的计算，细菌的绝对数量仅减少了一个数量级，仍远高于10^5CFU/g组织。 3.相比无菌纱布治疗，负压创面疗法能够降低金葡菌agrA的表达。 4.负压创面疗法能够显著减少eap, spa和hla的表达，其随时间变化具有明显减少趋势；组织中上述毒力因子含量低于无菌纱布治疗，随治疗时间延长而显著减少。 5.在感染早期，负压创面疗法一方面能够增加IL-1β和IL-8的表达，从调控上游激活固有免疫反应；另一方面抑制TNF-α的过度表达，避免出现过度炎症反应，降低自身损害。 6.负压创面疗法能够有效促进创面组织内中性粒细胞聚集，尤其在细菌侵润的创面浅表层中大量聚集。结论 1.负压创面疗法通过改变细菌的外部环境，从而影响菌群形态、增殖程度及侵袭深度等感染行为。 2.负压创面治疗一定程度上减少了组织内细菌的数量，但不是控制感染的主要作用机制。 3.负压作用下抑制了金葡菌群体感应agr系统的活性。 4.负压环境与agr系统的抑制使毒力因子表达减少；充分引流的协同作用使组织内毒力因子含量进一步减少。 5.负压创面疗法能够增强局部固有免疫反应，遏制感染扩大和蔓延。
[Abstract]:Research background
Infection in the field of Department of orthopedics has long been a difficult problem to be solved in the field of Department of orthopedics. Early research suggests that infection is a complex process of interaction between the bacteria and the host environment; the pathogenicity of Staphylococcus aureus is not only related to the quantity, but also depends on the state of bacterial flora and the expression of the virulence factor. Negative pressure wound therapy is a therapeutic method for effective treatment of infectious soft tissue injury. Negative pressure drainage can effectively control infection without reducing the number of bacteria, but its internal mechanism is not clear. To study the effect of negative pressure wound therapy on Staphylococcus aureus infection, quorum induction agr system, virulence and the mechanism of the body's inherent immune system, and to deepen the understanding and theory of bacterial infection control.
objective
1. to investigate the change of bacterial infection and the changes in the number of immediate bacteria in the tissue under the effect of negative pressure wound therapy; 2. to explore the effect of negative pressure drainage on the upriver induction system of Staphylococcus aureus, and the changes in the expression and content of the virulence factors; 3. the study of the chemotaxis aggregation of the inherent immune cells through the expression of the inflammatory factors. Objective to explore the influence of negative pressure wound therapy on the innate immune system.
Method
1. to establish an animal model of Staphylococcus aureus infection and open injury of soft tissue, through the contrast of conventional sterile gauze and negative pressure wound therapy, using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and laser confocal microscopy (LSCM) to analyze the morphology of bacteria in the surface and tissue of the wound surface, the depth and quantity of invasion, and evaluate the bacterial infection behavior. Change.
2. using the above experimental model and method, the activity of the quorum induction agr system of Staphylococcus aureus was evaluated by real time fluorescence quantitative PCR (Real-time PCR) and Western blot analysis (Western Blot), and the expression and content of the virulence factor were detected.
3. the expression of inflammatory factors IL-1 beta, IL-8 and TNF- alpha in the wound tissue was detected, and the specimens were examined by pathological examination, and the quantity of neutrophils aggregation in different tissue depth was quantitatively analyzed.
Result
1. compared with the treatment of sterile gauze, the surface of the wound surface and the bacteria in the tissue were distributed in the surface of the wound and the time of treatment was prolonged, and the Staphylococcus aureus scattered in the microbacteria group, and the proliferation was inactive, and the invasion depth of the bacteria decreased significantly.
2. the number of bacteria in the two groups was 141.9 + 15.4% and 34.6 + 5.5% (P < 0.01) before the treatment of Day8 with sterile gauze treatment and negative pressure wound treatment, but the absolute number of bacteria was only reduced by one order of magnitude according to the relative ratio, still far higher than that of the 10^5CFU/g group.
3. compared with sterile gauze treatment, negative pressure wound therapy can reduce the expression of Staphylococcus aureus agrA.
4. negative pressure wound therapy could significantly reduce the expression of EAP, spa and HLA, which decreased with time, and the content of the virulence factor in the tissue was lower than that of sterile gauze, and decreased significantly with the time of treatment.
5. in the early stage of infection, the negative pressure wound therapy can increase the expression of IL-1 beta and IL-8 on the one hand, activate the inherent immune response from the upstream and regulate the upstream. On the other hand, it inhibits the overexpression of TNF- alpha, avoids the excessive inflammatory reaction and reduces the self damage.
6. negative pressure wound therapy can effectively promote the aggregation of neutrophils in wound tissues, especially in the superficial layer of bacteria invaded wound.
conclusion
1. negative pressure wound therapy can affect the bacterial morphology, proliferation and invasion depth by changing the external environment of bacteria.
2. negative pressure wound treatment reduced the number of bacteria in the tissue to some extent, but it was not the main mechanism of infection control.
3. under negative pressure, the activity of Staphylococcus aureus quorum sensing agr system was inhibited.
4. the negative pressure environment and inhibition of agr system reduced the expression of virulence factors, and the synergistic effect of adequate drainage reduced the virulence factors in tissues.
5. negative pressure wound therapy can enhance the local innate immune response and contain the spread and spread of infection.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R641

【参考文献】