ALA-PDT对铜绿假单胞菌生物膜及其QS系统的影响的实验研究

发布时间：2018-04-08 10:51

本文选题：光动力疗法　切入点：铜绿假单胞菌　出处：《第三军医大学》2017年硕士论文

【摘要】：1背景与目的氨基酮戊酸-光动力疗法(δ-Aminolevulinic acid photodynamic therapy,ALA-PDT)被认为是一种非常有前景的新的消除微生物感染的治疗方法。PDT因它良好的疗效目前已经被广泛用于某些感染性皮肤病的治疗,如痤疮、病毒疣等。此外,一些研究发现PDT对于细菌真菌及其生物膜(Biofilm,BF)的感染同样有效,包括金黄色葡萄球菌、表皮葡萄球菌、铜绿假单胞菌、白色念珠菌及一些耐药菌(如耐甲氧西林金黄色葡萄球菌)等。然而,很少有PDT对生物膜结构、功能以及其中作用的机制的研究。群体感应(quorum sensing,QS)系统是铜绿假单胞菌(Pseudomonas aeruginosa,PA)生物膜形成和产生毒力因子的最重要的调节系统,而PDT对铜绿假单胞菌生物膜相关研究中对QS系统及相关基因的影响从未被报道过。本研究通过建立铜绿假单胞菌体外生物膜模型,探索ALA-PDT对铜绿假单胞菌杀灭作用及对其生物膜清除作用及机制。为我们临床应用ALA-PDT治疗铜绿假单胞菌相关感染提供实验依据。2方法1.建立铜绿假单胞菌体外生物膜模型,不同浓度的ALA、单纯照光、ALA-PDT分别根据分组要求进行相应的处理;2.用激光共聚焦显微镜(Confocal laser scanning microscopy,CLSM)检测ALA在铜绿假单胞菌体内的转化;3.用XTT活菌定量实验检测ALA-PDT对铜绿假单胞菌生物膜中细菌的杀灭作用;4.通过对铜绿假单胞菌生物膜的荧光染色,使用CLSM观察ALA-PDT对生物膜中活菌的杀灭作用及对生物膜三维结构的影响;5.使用扫描电镜(Scanning electron microscopy,SEM)观察ALA-PDT对铜绿假单胞菌生物膜中细菌及生物膜结构和功能的影响;6.毒力因子检测试验研究ALA-PDT对绿脓菌素和弹性蛋白酶分泌的影响;7.通过实时定量PCR(q RT-PCR)检测基因的表达,研究ALA-PDT对毒力因子调控基因(phz H、Las B)与QS相关基因(las I、las R、rhl I、rhl R)表达的影响。3结果1.ALA孵育组在CLSM下可见大量砖红色荧光,而对照组在CLSM下未见无明显荧光;2.XTT活菌定量实验提示,ALA-PDT能有效杀灭铜绿假单胞菌生物膜中的活细菌,这种杀灭作用与ALA的浓度呈正相关,单纯ALA孵育、单纯照光组与对照组无明显差异,不能产生光动力效应;3.从CLSM和SEM的观察结果看出,ALA-PDT对铜绿假单胞菌生物膜的清除作用与ALA的浓度呈正相关,且ALA-PDT能明显杀灭生物膜中的活细菌,同时破坏生物膜的三维结构,单纯ALA孵育、单纯照光组与对照组无明显差异,不能产生光动力效应;4.从毒力因子检测试验看出,ALA-PDT能显著抑制铜绿假单胞菌毒力因子的分泌,这种抑制作用与ALA的浓度呈正相关,单纯ALA孵育、单纯照光组与对照组无明显差异,不能对毒力因子的分泌产生抑制效应;5.从q RT-PCR试验结果看出,单纯ALA孵育和红光照射时,铜绿假单胞菌毒力因子调控基因和QS相关调控基因的表达无明显变化,只有ALA和红光照射同时存在的情况下,才能发挥PDT作用,ALA-PDT能明显降低QS系统相关基因的表达,这种对基因表达调控的抑制效应与ALA的浓度呈正比。4结论我们的研究初步阐明了ALA-PDT清除铜绿假单胞菌生物膜的作用及其相关机制。ALA-PDT产生的单线态氧能够产生毒性效应破坏靶细菌的细胞膜、细胞器、细胞核,同时破坏了QS系统的基因和蛋白,这种对QS系统的作用进一步破坏了生物膜,同时减少了生物膜的复发率。我们的研究结果表明,ALA-PDT可以有效的杀灭体外的铜绿假单胞菌及其生物膜,为我们在临床中使用PDT治疗铜绿假单胞菌相关感染提供了进一步的实验依据。PDT是目前对于耐药性或难治性细菌感染的最有前景的治疗方法之一,需要我们更加深入的进行研究。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R751.05

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