孟加拉红介导的光动力疗法对牙龈卟啉单胞菌抑制作用的研究

发布时间：2019-06-21 07:01

【摘要】：目的：光动力疗法（PDT）是光敏剂和特定波长激光之间的非热能光化学反应，在反应的过程中会产生单线态氧及氧自由基，从而对细胞产生不可逆的毒性作用。本研究通过在体外环境下利用菌悬液及构建细菌生物膜模型，初步检测及观察RB介导的PDT（RB-PDT）对牙龈卟啉单胞菌（P.g）的抑制作用，从而为RB-PDT预防和治疗牙周炎的临床应用提供理论及实验依据。方法：（1）应用不同浓度的RB在波长为490nm的LED光源激发作用下，观察其对P.g菌悬液的抑制效果。实验分成两组：对照组（0.9%生理盐水）和PDT组（RB浓度分别为50μM，20μM，10μM，5μM，1μM，共培养5分钟，LED灯光照3分钟）。处理完成后采用十倍稀释法稀释菌悬液后继续培养48小时。菌落计数法得出原菌液的菌落形成单位，，并进行统计分析。（2）利用96孔板建立P.g的48小时成熟生物膜模型，采用MTT法检测RB-PDT组（50μM），单纯光照组，单纯RB组及生理盐水组作用后P.g生物膜的吸光度值，比较各组之间的差异；（3）使用激光共聚焦专用培养皿建立P.g的48小时成熟生物膜模型，采用SYT09/PI荧光染色剂使细菌着色，利用激光共聚焦显微镜（CLSM）观察各药物处理组（RB-PDT组（50μM），单纯光照组，单纯RB组及生理盐水组）生物膜中细菌染色情况及死菌活菌比例。结果：（1）菌落平板计数法显示RB-PDT组(RB浓度分别为50μM，20μM，10μM，5μM，1μM)与生理盐水对照组相比，P.g菌落数（CFU/ml）有显著减少（P＜0.05），且五个不同浓度的RB-PDT组之间相互比较发现P.g的菌落数随着RB浓度的增加而减少；（2）MTT检测结果显示RB-PDT组（50μM）与单纯光照组，单纯RB组及生理盐水组比较差异均有显著性（P＜0.05），而单纯光照组，单纯RB组及生理盐水组之间差异无显著性（P0.05）；（3）CLSM图像显示RB-PDT组可见P.g菌体染色以红色（死菌）荧光为主，仅少量菌体染色为绿色（活菌）荧光，同一视图叠加后以红色荧光为主，而单纯光照组，单纯RB组及生理盐水组镜下菌体菌体染色以绿色（活菌）荧光为主，仅少量菌体染色为红色（死菌）荧光，同一视图叠加后以绿色荧光为主。结论：（1）RB-PDT对P.g菌悬液具有明显的抑制和破坏作用，且RB-PDT的抑制作用有浓度相关性；（2）P.g的菌斑生物膜构建成功，RB-PDT对P.g菌斑生物膜具有明显的抑制和破坏作用；单纯光照组和单纯RB组对P.g生物膜无明显抑菌作用。
[Abstract]:Aim: photodynamic therapy (PDT) is a non-thermal photochemical reaction between photosensitizer and laser at a specific wavelength. Singlet oxygen and oxygen free radicals are produced in the process of reaction, which can produce irreversible toxicity to cells. In this study, the inhibitory effect of RB-mediated PDT (RB-PDT) on Porphyromonas gums (P.G) was detected and observed by using bacterial suspension and bacterial biofilm model in vitro, so as to provide theoretical and experimental basis for the clinical application of RB-PDT in the prevention and treatment of periodontitis. Methods: (1) the inhibitory effect of different concentrations of RB on P.g suspension was observed under the excitation of LED light source with wavelength of 490nm. The experiment was divided into two groups: control group (0.9% saline) and PDT group (RB concentration 50 渭 M, 20 渭 M, 10 渭 M, 5 渭 M, 1 渭 M, co-culture for 5 minutes, LED lighting for 3 minutes). After the treatment, the bacteria suspension was diluted by ten times dilution method and cultured for 48 hours. The colony forming unit of the original bacteria solution was obtained by colony counting method, and the statistical analysis was carried out. (2) the 48-hour mature biofilm model of P.G was established by 96-well plate. The absorbance values of P.G biofilm in RB-PDT group (50 渭 M), irradiation group, RB group and saline group were measured by MTT method, and the differences among the three groups were compared. (3) the 48-hour mature biofilm model of P.G was established by laser confocal petri dish. The bacteria were stained by SYT09/PI fluorescent staining. The bacterial staining and the proportion of dead bacteria in the biofilm of each drug treatment group (RB-PDT group (50 渭 M), simple light group, RB group and saline group) were observed by laser confocal microscope (CLSM). Results: (1) compared with saline control group, the colony number (CFU/ml) of RB-PDT group (RB concentration 50 渭 M, 20 渭 M, 10 渭 M, 5 渭 M, 1 渭 M) was significantly lower than that of saline control group (P < 0.05). Compared with the five different concentrations of RB-PDT group, the colony number of P.G decreased with the increase of RB concentration. (2) the results of MTT showed that there was significant difference between RB-PDT group (50 渭 M) and simple light group, RB group and saline group, but there was no significant difference between light group, RB group and saline group (P 0.05). (3) CLSM images showed that the staining of P.G cells in RB-PDT group was mainly red (dead bacteria) fluorescence, only a small number of bacteria were stained with green (living bacteria) fluorescence, and only a small amount of bacteria were stained with red fluorescence after superposition of the same view, while in the simple light group, simple RB group and saline group, the staining of P.G bacteria was mainly green (living bacteria) fluorescence, and only a small amount of bacteria were stained with red (dead bacteria) fluorescence. The same view is superimposed with green fluorescence. Conclusion: (1) RB-PDT has obvious inhibitory and destructive effect on P.G. Bacteria suspension, and the inhibitory effect of RB-PDT is concentration-dependent. (2) the plaque biofilm of P.G was constructed successfully, and RB-PDT had obvious inhibitory and destructive effect on P.G biofilm, while the irradiation group and RB group had no obvious bacteriostatic effect on P.G biofilm.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4

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