發(fā)布時間：2018-05-11 05:16

  本文選題：白色念珠菌 + 生物膜��； 參考：《復旦大學》2014年碩士論文

【摘要】：目的：本實驗通過體外建立白色念珠菌(Candida albicans)生物膜模型,在此基礎上探索5-氨基酮戊酸光動力療法(ALA-PDT)對白色念珠菌生物膜活性及結構上的抑制效果,尋找最佳ALA孵育時間、最佳ALA用藥濃度、最佳光照劑量等光動力參數(shù),為今后進一步將ALA-PDT推廣到臨床用于治療口腔白色念珠菌感染性疾病提供理論依據(jù)。方法：1.建立白色念珠菌生物膜體外模型：選擇白色念珠菌標準株ATCC90028,分別用96孔細胞培養(yǎng)板和玻璃圓皿培養(yǎng)白色念珠菌生物膜。結合結晶紫染色、熒光雙染色的方法,利用普通光學顯微鏡及激光共聚焦掃描顯微鏡(confocal laser scanning microscopy, CLSM)對白色念珠菌生物膜的形成過程及結構進行超微結構的觀察與定性分析,并采用甲基四氮鹽(XTT)減低法對生物膜的生長動力學進行定量分析,為之后的實驗奠定模型基礎。2.研究ALA對白色念珠菌生物膜的最佳作用參數(shù)：運用熒光分光光度儀檢測ALA在生物膜內(nèi)的吸收和轉化為PpIX的情況,研究ALA在白色念珠菌生物膜內(nèi)的最佳參數(shù),確定最佳ALA孵育時間和最佳ALA用藥濃度,為之后的實驗提供理論依據(jù)。3.評估ALA-PDT對白色念珠菌生物膜的影響：本部分實驗建立在前兩部分研究的基礎上,利用CLSM檢測ALA-PDT過程中不同光照劑量對白色念珠菌生物膜活性的抑制作用,計算殺菌率的變化,并定量分析ALA-PDT對生物膜結構的破壞效果,計算生物膜平均厚度的變化,最終確定最佳光照劑量。結果：1. 白色念珠菌生物膜的體外模型建立白色念珠菌ATCC90028培養(yǎng)48h后,經(jīng)結晶紫染色及光學顯微鏡觀察,可見生物膜緊密附著于載體底部,不被外力吹打和PBS洗滌所破壞。經(jīng)熒光雙染色及CLSM觀察,可見典型的生物膜特征：具有一定厚度的主要由細胞外基質(zhì)組成的緊密膜狀結構。XTT減低法分析顯示48h的生物膜生長基本飽和,進入了成熟的穩(wěn)定狀態(tài)。2.ALA對白色念珠菌生物膜的最佳作用參數(shù)白色念珠菌生物膜與ALA孵育后,CLSM下可見PpIX磚紅色熒光,說明白色念珠菌生物膜能夠吸收ALA并轉化為PpIX。通過根據(jù)ALA孵育時間和ALA用藥濃度的交叉實驗發(fā)現(xiàn),熒光強度的變化具有一定的時間依賴性,但不具有濃度依賴性。本實驗最終以ALA孵育時間為5h、ALA用藥濃度為15mM作為ALA對白色念珠菌生物膜的最佳作用參數(shù)。3. ALA-PDT對生物膜的抑制效果經(jīng)過不同劑量的紅光照射后,CLSM雙染色觀察到死菌數(shù)量隨光照劑量的增加而增加,生物膜的結構也隨光照劑量的增加而疏松,生物膜的平均厚度隨光照劑量的增加而減少。當光劑量達300J/cm2時,殺菌率接近80%,生物膜平均厚度縮小超過一半。結論：1.白色念珠菌ATCC90028可以經(jīng)體外培養(yǎng)48h形成穩(wěn)定的白色念珠菌生物膜模型。2.白色念珠菌生物膜能夠吸收外源性ALA并轉化為具有光敏性的物質(zhì)PpIX。3. ALA-PDT能夠對白色念珠菌生物膜內(nèi)的細胞活性起抑制作用,并且對生物膜結構本身也一定的破壞作用。4. ALA-PDT作用于體外白色念珠菌生物膜的最佳作用參數(shù)：ALA最佳孵育時間為5h,ALA最佳用藥濃度為15mM, ALA-PDT最佳光照劑量為300J/cm2。
[Abstract]:Objective: To explore the inhibitory effect of 5- aminolevulinic acid photodynamic therapy (ALA-PDT) on the bioactivity and structure of Candida albicans by establishing the Candida albicans biomembrane model in vitro, and to find the best ALA incubation time, the best ALA concentration, the best light dose and other photodynamic parameters. In the future, ALA-PDT will be further extended to the clinic for the treatment of oral Candida albicans infection disease. Methods: 1. the model of Candida albicans biofilm was established: the standard strain ATCC90028 of Candida albicans was selected, and the Candida albicans biofilm was cultured with 96 pore cell culture plate and glass dish. The process and structure of the biofilm of Candida albicans were observed and qualitatively analyzed by common optical microscope and confocal laser scanning microscopy (CLSM), and the growth kinetics of the biofilm was determined by methyl four nitrogen salt (XTT) reduction method. Quantitative analysis, the best parameters of ALA for Candida albicans biofilm were studied by.2.. The best parameters of ALA in the biofilm of Candida albicans were studied by fluorescence spectrophotometer, and the best time of ALA incubation and the best concentration of ALA were studied. The effect of ALA-PDT on the biofilm of Candida albicans was evaluated based on.3.. Based on the first two parts of the study, this experiment used CLSM to detect the inhibitory effect of different light doses on the activity of Candida albicans during the process of ALA-PDT, to calculate the change of bactericidal rate, and to analyze the quantitative analysis of ALA-PDT pairs. The destruction effect of biofilm structure, calculate the change of average thickness of biofilm, and finally determine the best light dose. Results: 1. Candida albicans biofilm model in vitro model of Candida albicans ATCC90028 culture 48h, crystal violet staining and optical microscope observation, it can be seen that biofilm tightly attached to the bottom of the carrier, not to be blown by external force. The characteristics of the typical biofilm were observed by fluorescence double staining and CLSM observation. The analysis of the compact membranous structure with a certain thickness mainly composed of extracellular matrix showed that the growth of the biofilm of 48h was basically saturated, and the optimum effect of.2.ALA on the biofilm of Candida albicans was entered into the mature state of.2.ALA. After incubation of the Candida albicans biofilm with ALA, the red fluorescence of PpIX brick can be seen under CLSM. It shows that the Candida albicans biofilm can absorb ALA and transform into PpIX. through the cross experiment based on the incubation time of ALA and the concentration of ALA drugs. It is found that the change of fluorescence intensity has a certain time dependence, but it is not dependent on concentration. This experiment is the most important. At the end of ALA incubation time was 5h, the concentration of ALA was 15mM as the best parameter of ALA on the biofilm of Candida albicans. The inhibitory effect of.3. ALA-PDT on the biofilm was irradiated in different doses of red light. The number of dead bacteria increased with the increase of light dose, and the structure of the biofilm was also increased with the increase of light dose. The average thickness of the biofilm decreases with the increase of light dose. When the light dose reaches 300J/cm2, the bactericidal rate is close to 80%, and the average thickness of the biofilm is reduced by more than half. Conclusion: 1. Candida albicans can be cultured in vitro to form a stable Candida albicans biofilm model,.2., to absorb the external Candida albicans biofilm. Derived ALA and transformed into a photosensitive substance PpIX.3. ALA-PDT can inhibit the cell activity in the Candida albicans biofilm, and the damage to the biofilm structure itself is also the best parameter for the effect of.4. ALA-PDT on the Candida albicans biofilm in vitro: the best incubation time of ALA is 5h, and the best medication for ALA The optimum light dose was 15mM and ALA-PDT was 300J/cm2..

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R780.2

【共引文獻】

相關博士學位論文 前1條

1 趙占娟;新型光敏抗菌藥物的研究[D];北京協(xié)和醫(yī)學院;2014年

相關碩士學位論文 前2條

1 羅雪晴;孟加拉紅介導的光動力療法對牙齦卟啉單胞菌抑制作用的研究[D];南昌大學;2014年

2 靳曉蘭;光動力療法聯(lián)合EDTA應用于根管消毒的實驗研究[D];天津醫(yī)科大學;2014年

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本文編號：1872551