發(fā)布時間：2019-06-21 07:01

【摘要】：目的： 光動力療法（PDT）是光敏劑和特定波長激光之間的非熱能光化學(xué)反應(yīng)，在反應(yīng)的過程中會產(chǎn)生單線態(tài)氧及氧自由基，從而對細(xì)胞產(chǎn)生不可逆的毒性作用。本研究通過在體外環(huán)境下利用菌懸液及構(gòu)建細(xì)菌生物膜模型，初步檢測及觀察RB介導(dǎo)的PDT（RB-PDT）對牙齦卟啉單胞菌（P.g）的抑制作用，從而為RB-PDT預(yù)防和治療牙周炎的臨床應(yīng)用提供理論及實驗依據(jù)。 方法： （1）應(yīng)用不同濃度的RB在波長為490nm的LED光源激發(fā)作用下，觀察其對P.g菌懸液的抑制效果。實驗分成兩組：對照組（0.9%生理鹽水）和PDT組（RB濃度分別為50μM，20μM，10μM，5μM，1μM，共培養(yǎng)5分鐘，LED燈光照3分鐘）。處理完成后采用十倍稀釋法稀釋菌懸液后繼續(xù)培養(yǎng)48小時。菌落計數(shù)法得出原菌液的菌落形成單位，，并進(jìn)行統(tǒng)計分析。 （2）利用96孔板建立P.g的48小時成熟生物膜模型，采用MTT法檢測RB-PDT組（50μM），單純光照組，單純RB組及生理鹽水組作用后P.g生物膜的吸光度值，比較各組之間的差異； （3）使用激光共聚焦專用培養(yǎng)皿建立P.g的48小時成熟生物膜模型，采用SYT09/PI熒光染色劑使細(xì)菌著色，利用激光共聚焦顯微鏡（CLSM）觀察各藥物處理組（RB-PDT組（50μM），單純光照組，單純RB組及生理鹽水組）生物膜中細(xì)菌染色情況及死菌活菌比例。 結(jié)果： （1）菌落平板計數(shù)法顯示RB-PDT組(RB濃度分別為50μM，20μM，10μM，5μM，1μM)與生理鹽水對照組相比，P.g菌落數(shù)（CFU/ml）有顯著減少（P＜0.05），且五個不同濃度的RB-PDT組之間相互比較發(fā)現(xiàn)P.g的菌落數(shù)隨著RB濃度的增加而減少； （2）MTT檢測結(jié)果顯示RB-PDT組（50μM）與單純光照組，單純RB組及生理鹽水組比較差異均有顯著性（P＜0.05），而單純光照組，單純RB組及生理鹽水組之間差異無顯著性（P0.05）； （3）CLSM圖像顯示RB-PDT組可見P.g菌體染色以紅色（死菌）熒光為主，僅少量菌體染色為綠色（活菌）熒光，同一視圖疊加后以紅色熒光為主，而單純光照組，單純RB組及生理鹽水組鏡下菌體菌體染色以綠色（活菌）熒光為主，僅少量菌體染色為紅色（死菌）熒光，同一視圖疊加后以綠色熒光為主。 結(jié)論： （1）RB-PDT對P.g菌懸液具有明顯的抑制和破壞作用，且RB-PDT的抑制作用有濃度相關(guān)性； （2）P.g的菌斑生物膜構(gòu)建成功，RB-PDT對P.g菌斑生物膜具有明顯的抑制和破壞作用；單純光照組和單純RB組對P.g生物膜無明顯抑菌作用。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.4

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