本文選題：感染 + 負(fù)壓創(chuàng)面療法�。� 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：研究背景 創(chuàng)面感染一直是骨科領(lǐng)域長期致力解決的難題。前期研究認(rèn)為：感染是細(xì)菌與宿主體內(nèi)環(huán)境相互作用的復(fù)雜過程；金葡菌致病力不僅與數(shù)量有關(guān)，更取決于細(xì)菌菌群狀態(tài)和毒力因子表達(dá)。而且病原菌會(huì)適應(yīng)環(huán)境變化調(diào)節(jié)相應(yīng)的致病基因表達(dá)模式。負(fù)壓創(chuàng)面療法是一種能夠有效治療感染性軟組織損傷的治療方法，通過負(fù)壓引流作用可在不減少細(xì)菌數(shù)量的情況下，顯著控制感染，但其內(nèi)在機(jī)制尚不明確。本研究擬采用金黃色葡萄球菌感染軟組織損傷動(dòng)物模型，通過研究負(fù)壓創(chuàng)面療法對金葡菌感染行為、群體感應(yīng)agr系統(tǒng)、毒力狀態(tài)以及機(jī)體固有免疫系統(tǒng)的作用機(jī)制，深化細(xì)菌性感染控制的認(rèn)識與理論。 目的 1.探討負(fù)壓創(chuàng)面療法作用下細(xì)菌感染行為的改變與組織內(nèi)即時(shí)細(xì)菌數(shù)量的變化趨勢；2.探討負(fù)壓引流作用對金葡菌上游群體感應(yīng)系統(tǒng)的影響，以及毒力因子表達(dá)與含量的變化；3.通過對炎性因子表達(dá)，固有免疫細(xì)胞趨化聚集的研究，探索負(fù)壓創(chuàng)面療法對機(jī)體固有免疫系統(tǒng)的影響。 方法 1.建立金葡菌感染軟組織開放性損傷動(dòng)物模型,通過常規(guī)無菌紗布治療與負(fù)壓創(chuàng)面療法的對照，應(yīng)用掃描電鏡（SEM）、透射電鏡（TEM）、激光共聚焦顯微鏡（LSCM）等方法分析創(chuàng)面表面及組織內(nèi)菌群形態(tài)、侵襲深度及數(shù)量變化，評估細(xì)菌感染行為的變化。 2.應(yīng)用上述實(shí)驗(yàn)?zāi)Ｐ团c方法，通過實(shí)時(shí)熒光定量PCR（Real-time PCR）、免疫印跡分析（Western Blot）評估金葡菌群體感應(yīng)agr系統(tǒng)活性，并檢測毒力因子的表達(dá)及含量。 3.檢測創(chuàng)面組織內(nèi)炎性因子IL-1β、IL-8和TNF-α的表達(dá)，對標(biāo)本進(jìn)行病理學(xué)檢查，并定量分析不同組織深度內(nèi)中性粒細(xì)胞聚集的數(shù)量。 結(jié)果 1.相比無菌紗布治療，，負(fù)壓創(chuàng)面療法作用下創(chuàng)面表面和組織內(nèi)細(xì)菌均呈散在分布，且隨治療時(shí)間延長逐步明顯；微菌群中的金葡菌散在分布，增生不活躍；同時(shí)細(xì)菌侵襲深度顯著減低。 2.無菌紗布治療與負(fù)壓創(chuàng)面療法兩組在治療day8時(shí)，創(chuàng)面細(xì)菌數(shù)量分別為治療前的141.9±15.4%和34.6±5.5%(P＜0.01)；但根據(jù)相對比值的計(jì)算，細(xì)菌的絕對數(shù)量僅減少了一個(gè)數(shù)量級，仍遠(yuǎn)高于10^5CFU/g組織。 3.相比無菌紗布治療，負(fù)壓創(chuàng)面療法能夠降低金葡菌agrA的表達(dá)。 4.負(fù)壓創(chuàng)面療法能夠顯著減少eap, spa和hla的表達(dá)，其隨時(shí)間變化具有明顯減少趨勢；組織中上述毒力因子含量低于無菌紗布治療，隨治療時(shí)間延長而顯著減少。 5.在感染早期，負(fù)壓創(chuàng)面療法一方面能夠增加IL-1β和IL-8的表達(dá)，從調(diào)控上游激活固有免疫反應(yīng)；另一方面抑制TNF-α的過度表達(dá)，避免出現(xiàn)過度炎癥反應(yīng)，降低自身損害。 6.負(fù)壓創(chuàng)面療法能夠有效促進(jìn)創(chuàng)面組織內(nèi)中性粒細(xì)胞聚集，尤其在細(xì)菌侵潤的創(chuàng)面淺表層中大量聚集。 結(jié)論 1.負(fù)壓創(chuàng)面療法通過改變細(xì)菌的外部環(huán)境，從而影響菌群形態(tài)、增殖程度及侵襲深度等感染行為。 2.負(fù)壓創(chuàng)面治療一定程度上減少了組織內(nèi)細(xì)菌的數(shù)量，但不是控制感染的主要作用機(jī)制。 3.負(fù)壓作用下抑制了金葡菌群體感應(yīng)agr系統(tǒng)的活性。 4.負(fù)壓環(huán)境與agr系統(tǒng)的抑制使毒力因子表達(dá)減少；充分引流的協(xié)同作用使組織內(nèi)毒力因子含量進(jìn)一步減少。 5.負(fù)壓創(chuàng)面療法能夠增強(qiáng)局部固有免疫反應(yīng)，遏制感染擴(kuò)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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R641

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