本文選題：光動(dòng)力療法　切入點(diǎn)：銅綠假單胞菌　出處：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：1背景與目的氨基酮戊酸-光動(dòng)力療法(δ-Aminolevulinic acid photodynamic therapy,ALA-PDT)被認(rèn)為是一種非常有前景的新的消除微生物感染的治療方法。PDT因它良好的療效目前已經(jīng)被廣泛用于某些感染性皮膚病的治療,如痤瘡、病毒疣等。此外,一些研究發(fā)現(xiàn)PDT對(duì)于細(xì)菌真菌及其生物膜(Biofilm,BF)的感染同樣有效,包括金黃色葡萄球菌、表皮葡萄球菌、銅綠假單胞菌、白色念珠菌及一些耐藥菌(如耐甲氧西林金黃色葡萄球菌)等。然而,很少有PDT對(duì)生物膜結(jié)構(gòu)、功能以及其中作用的機(jī)制的研究。群體感應(yīng)(quorum sensing,QS)系統(tǒng)是銅綠假單胞菌(Pseudomonas aeruginosa,PA)生物膜形成和產(chǎn)生毒力因子的最重要的調(diào)節(jié)系統(tǒng),而PDT對(duì)銅綠假單胞菌生物膜相關(guān)研究中對(duì)QS系統(tǒng)及相關(guān)基因的影響從未被報(bào)道過(guò)。本研究通過(guò)建立銅綠假單胞菌體外生物膜模型,探索ALA-PDT對(duì)銅綠假單胞菌殺滅作用及對(duì)其生物膜清除作用及機(jī)制。為我們臨床應(yīng)用ALA-PDT治療銅綠假單胞菌相關(guān)感染提供實(shí)驗(yàn)依據(jù)。2方法1.建立銅綠假單胞菌體外生物膜模型,不同濃度的ALA、單純照光、ALA-PDT分別根據(jù)分組要求進(jìn)行相應(yīng)的處理;2.用激光共聚焦顯微鏡(Confocal laser scanning microscopy,CLSM)檢測(cè)ALA在銅綠假單胞菌體內(nèi)的轉(zhuǎn)化;3.用XTT活菌定量實(shí)驗(yàn)檢測(cè)ALA-PDT對(duì)銅綠假單胞菌生物膜中細(xì)菌的殺滅作用;4.通過(guò)對(duì)銅綠假單胞菌生物膜的熒光染色,使用CLSM觀察ALA-PDT對(duì)生物膜中活菌的殺滅作用及對(duì)生物膜三維結(jié)構(gòu)的影響;5.使用掃描電鏡(Scanning electron microscopy,SEM)觀察ALA-PDT對(duì)銅綠假單胞菌生物膜中細(xì)菌及生物膜結(jié)構(gòu)和功能的影響;6.毒力因子檢測(cè)試驗(yàn)研究ALA-PDT對(duì)綠膿菌素和彈性蛋白酶分泌的影響;7.通過(guò)實(shí)時(shí)定量PCR(q RT-PCR)檢測(cè)基因的表達(dá),研究ALA-PDT對(duì)毒力因子調(diào)控基因(phz H、Las B)與QS相關(guān)基因(las I、las R、rhl I、rhl R)表達(dá)的影響。3結(jié)果1.ALA孵育組在CLSM下可見(jiàn)大量磚紅色熒光,而對(duì)照組在CLSM下未見(jiàn)無(wú)明顯熒光;2.XTT活菌定量實(shí)驗(yàn)提示,ALA-PDT能有效殺滅銅綠假單胞菌生物膜中的活細(xì)菌,這種殺滅作用與ALA的濃度呈正相關(guān),單純ALA孵育、單純照光組與對(duì)照組無(wú)明顯差異,不能產(chǎn)生光動(dòng)力效應(yīng);3.從CLSM和SEM的觀察結(jié)果看出,ALA-PDT對(duì)銅綠假單胞菌生物膜的清除作用與ALA的濃度呈正相關(guān),且ALA-PDT能明顯殺滅生物膜中的活細(xì)菌,同時(shí)破壞生物膜的三維結(jié)構(gòu),單純ALA孵育、單純照光組與對(duì)照組無(wú)明顯差異,不能產(chǎn)生光動(dòng)力效應(yīng);4.從毒力因子檢測(cè)試驗(yàn)看出,ALA-PDT能顯著抑制銅綠假單胞菌毒力因子的分泌,這種抑制作用與ALA的濃度呈正相關(guān),單純ALA孵育、單純照光組與對(duì)照組無(wú)明顯差異,不能對(duì)毒力因子的分泌產(chǎn)生抑制效應(yīng);5.從q RT-PCR試驗(yàn)結(jié)果看出,單純ALA孵育和紅光照射時(shí),銅綠假單胞菌毒力因子調(diào)控基因和QS相關(guān)調(diào)控基因的表達(dá)無(wú)明顯變化,只有ALA和紅光照射同時(shí)存在的情況下,才能發(fā)揮PDT作用,ALA-PDT能明顯降低QS系統(tǒng)相關(guān)基因的表達(dá),這種對(duì)基因表達(dá)調(diào)控的抑制效應(yīng)與ALA的濃度呈正比。4結(jié)論我們的研究初步闡明了ALA-PDT清除銅綠假單胞菌生物膜的作用及其相關(guān)機(jī)制。ALA-PDT產(chǎn)生的單線態(tài)氧能夠產(chǎn)生毒性效應(yīng)破壞靶細(xì)菌的細(xì)胞膜、細(xì)胞器、細(xì)胞核,同時(shí)破壞了QS系統(tǒng)的基因和蛋白,這種對(duì)QS系統(tǒng)的作用進(jìn)一步破壞了生物膜,同時(shí)減少了生物膜的復(fù)發(fā)率。我們的研究結(jié)果表明,ALA-PDT可以有效的殺滅體外的銅綠假單胞菌及其生物膜,為我們?cè)谂R床中使用PDT治療銅綠假單胞菌相關(guān)感染提供了進(jìn)一步的實(shí)驗(yàn)依據(jù)。PDT是目前對(duì)于耐藥性或難治性細(xì)菌感染的最有前景的治療方法之一,需要我們更加深入的進(jìn)行研究。
[Abstract]:1 background and objective topical 5-aminolevulinic acid photodynamic therapy (-Aminolevulinic acid photodynamic therapy 8, ALA-PDT) is considered to be a promising new treatment method for eliminating microorganisms infection due to its good curative effect of.PDT has been widely used in treatment of some infectious skin diseases such as acne, warts. In addition and some studies have found that PDT for bacteria and fungi and biofilm (Biofilm, BF) infection equally effective, including Staphylococcus aureus, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and some resistant bacteria (such as methicillin-resistant Staphylococcus aureus). However, there are few PDT on membrane structure study on the mechanism, function and the role of quorum sensing. (quorum sensing, QS) is Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) biofilm formation and virulence factors of the most important regulation System, and the related studies on Pseudomonas aeruginosa biofilm PDT effects on QS system and related gene has never been reported. In this study, through the establishment of Pseudomonas aeruginosa biofilm model in vitro, exploring the ALA-PDT of Pseudomonas aeruginosa and its killing effect on biofilm removal effect and mechanism. The establishment of 1. Pseudomonas aeruginosa in vitro bacteria biofilm model to provide experimental basis for.2 infection we clinical application of ALA-PDT treatment of Pseudomonas aeruginosa, different concentrations of ALA, pure light, ALA-PDT were respectively treated according to the grouping requirements; 2. by laser scanning confocal microscope (Confocal laser scanning microscopy, CLSM) detection of ALA transformation in Pseudomonas aeruginosa in 3.; with live XTT kill bacteria quantitative assay of ALA-PDT on bacterial biofilm of Pseudomonas aeruginosa in 4.; through the fluorescence of Pseudomonas aeruginosa biofilm to staining. The effect of ALA-PDT on the killing effect of live bacteria in the biofilm and the influence of the three-dimensional structure of biological membrane by using scanning electron microscopy (CLSM; 5. Scanning electron microscopy, SEM) to observe the effect of ALA-PDT on the structure and function of bacteria and biofilm of Pseudomonas aeruginosa biofilm; experimental study on detection of 6. virulence factors of Pseudomonas aeruginosa ALA-PDT and effect of elastase secretion; 7. by quantitative real-time PCR (Q RT-PCR) to detect the expression of genes, regulation of virulence factor gene of ALA-PDT (PHZ H, Las B) and QS (Las I, Las related gene R, RHL I, RHL R). The results of.3 expression in 1.ALA group were incubated in CLSM a large number of visible brick red fluorescence, while the control group had no obvious fluorescence was not found in CLSM; 2.XTT live bacteria quantitative experimental tips ALA-PDT can effectively kill bacteria, Pseudomonas aeruginosa biofilm, and ALA concentration was positively related to the killing effect, simple ALA incubation That simple irradiation group had no significant difference with the control group, can not produce photodynamic effect; 3. from the observation of CLSM and SEM. The results showed that the concentration of ALA-PDT was positively on Pseudomonas aeruginosa biofilm scavenging effects associated with ALA, and ALA-PDT can significantly kill live bacteria in the biofilm, biofilm with three-dimensional structure damage the simple ALA incubation, simple irradiation group had no significant difference with the control group, can not produce photodynamic effect; 4. virulence factors from the test showed that ALA-PDT can significantly inhibit the secretion of virulence factors of Pseudomonas aeruginosa, this inhibition concentration was positively correlated with ALA, simple ALA incubation, simple irradiation group the obvious difference with the control group, not on the secretion of virulence factors have inhibition effect; 5. from the Q RT-PCR test results show that pure ALA incubation and red light irradiation, Pseudomonas aeruginosa virulence factor genes and QS genes No change in the expression of ALA and red light irradiation, only exist at the same time, in order to play the role of PDT, ALA-PDT can significantly reduce the expression of genes related to QS system, and ALA inhibited the regulation of gene expression in the cell membrane, we study the proportional.4 conclusion illustrates the singlet oxygen.ALA-PDT ALA-PDT removal of P. Pseudomonas aeruginosa biofilm effect and mechanism of the toxic effects can destroy target bacterial cells, the nucleus, but also destroy the gene and protein of the QS system, the QS system further damage to the biofilm, while reducing the biofilm recurrence rate. Our results show that ALA-PDT of Pseudomonas aeruginosa biofilm and killing in vitro effectively and provide further experimental basis for us to use PDT in the clinical treatment of Pseudomonas aeruginosa infection. PDT is one of the most promising treatments for drug-resistant or refractory bacterial infections, and we need to study more deeply.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R751.05

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 譚楊;李鳳;成瓊輝;伍津津;雷霞;;慢性皮膚潰瘍172例臨床表現(xiàn)及難愈原因分析[J];創(chuàng)傷外科雜志;2016年05期

，

本文編號(hào)：1721286