【摘要】：研究背景與目的在醫(yī)療領域,皮膚軟組織感染仍然是亟需解決的難題。由感染導致的創(chuàng)面延遲愈合、不愈合,病情加重,住院時間延長等等給患者帶來了沉重的心理及經(jīng)濟負擔。臨床上,負壓創(chuàng)面療法被廣泛用于處理開放傷可能伴有感染的患者,且方法有效。然而,原因與機制并不完全清楚。有研究發(fā)現(xiàn)負壓環(huán)境下創(chuàng)面細菌數(shù)量會發(fā)生變化,這可能是負壓創(chuàng)面療法減輕感染的機制之一。而負壓環(huán)境下銅綠假單胞菌的細菌數(shù)量、毒素毒力等變化機制目前相關研究甚少。本研究采用銅綠假單胞菌感染軟組織損傷的模型,研究負壓環(huán)境對銅綠假單胞菌體外體內增殖、毒素分泌、毒力表達以及生物膜的相關作用及其機制。研究方法1.創(chuàng)建銅綠假單胞菌PAO1體外增殖模型,觀察負壓對細菌增殖的相關作用。應用銅綠假單胞菌PAO1侵染軟組織損傷動物模型,通過負壓創(chuàng)面療法與常規(guī)紗布治療的對照研究,采用多點取材培養(yǎng)法分析創(chuàng)面軟組織內細菌數(shù)量的變化趨勢及其原因。2.采用上述動物模型,通過酶聯(lián)免疫吸附法、濃硫酸-苔黑酚法、彈性蛋白-剛果紅法檢測創(chuàng)面軟組織內毒素含量,通過掃描電鏡、刀豆蛋白A染色等方法觀察創(chuàng)面生物膜的變化。3.采用上述動物模型,通過實時熒光定量PCR (Real-time PCR)法評估銅綠假單胞菌毒素及生物膜調控基因表達變化。結果1.負壓作用下銅綠假單胞菌在LB固體培養(yǎng)基上增殖24小時、48小時細菌生長直徑均小于非負壓組,p0.01。2.與常規(guī)紗布組相比,負壓創(chuàng)面療法治療下創(chuàng)面軟組織內細菌數(shù)量減少明顯,在治療的第四、六、八天軟組織內細菌數(shù)量較常規(guī)紗布治療組低,p0.01。但是兩種治療方法均沒能將創(chuàng)面細菌數(shù)量降低到臨床感染標準(10^5CFU/g組織)以下。3.與常規(guī)紗布組相比,負壓創(chuàng)面療法組創(chuàng)面軟組織內細菌外毒素A、鼠李糖脂、彈性蛋白酶等毒素含量相對較低。4.負壓創(chuàng)面療法能夠減少創(chuàng)面銅綠假單胞菌生物膜。5.負壓創(chuàng)面療法可以顯著抑制銅綠假單胞菌toxA, rhlA, LasB, Lasl, Rh lI等基因表達水平。結論1.負壓環(huán)境可以有效的抑制銅綠假單胞菌的增殖。負壓創(chuàng)面療法在一定程度上可以減少創(chuàng)面軟組織內細菌數(shù)量,且效果優(yōu)于常規(guī)紗布治療。2.負壓與引流的綜合作用可以減少創(chuàng)面軟組織內銅綠假單胞菌毒素的含量,且效果優(yōu)于常規(guī)紗布治療。負壓環(huán)境可以抑制創(chuàng)面上銅綠假單胞菌毒素調控基因、生物膜調控基因的表達水平。3.負壓創(chuàng)面療法減輕銅綠假單胞菌創(chuàng)面感染的機制有抑制創(chuàng)面細菌增殖,抽吸引流創(chuàng)面細菌、毒素,抑制銅綠假單胞菌毒素及生物膜調控基因表達。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R641

【共引文獻】

相關期刊論文 前1條

1 潘婷婷;李鏞;劉嘉琳;瞿洪平;;銅綠假單胞菌感應系統(tǒng)基因表達與毒力因子的相關性分析[J];內科理論與實踐;2015年04期

相關碩士學位論文 前1條

1 張美光;負壓封閉引流聯(lián)合含氧液沖洗治療慢性創(chuàng)面的臨床研究[D];南方醫(yī)科大學;2014年

，

本文編號：2213438