5-氨基酮戊酸光动力疗法（ALA-PDT）对白色念珠菌生物膜的影响

发布时间：2018-05-11 05:16

本文选题：白色念珠菌 + 生物膜　；参考：《复旦大学》2014年硕士论文

【摘要】：目的：本实验通过体外建立白色念珠菌(Candida albicans)生物膜模型,在此基础上探索5-氨基酮戊酸光动力疗法(ALA-PDT)对白色念珠菌生物膜活性及结构上的抑制效果,寻找最佳ALA孵育时间、最佳ALA用药浓度、最佳光照剂量等光动力参数,为今后进一步将ALA-PDT推广到临床用于治疗口腔白色念珠菌感染性疾病提供理论依据。方法：1.建立白色念珠菌生物膜体外模型：选择白色念珠菌标准株ATCC90028,分别用96孔细胞培养板和玻璃圆皿培养白色念珠菌生物膜。结合结晶紫染色、荧光双染色的方法,利用普通光学显微镜及激光共聚焦扫描显微镜(confocal laser scanning microscopy, CLSM)对白色念珠菌生物膜的形成过程及结构进行超微结构的观察与定性分析,并采用甲基四氮盐(XTT)减低法对生物膜的生长动力学进行定量分析,为之后的实验奠定模型基础。2.研究ALA对白色念珠菌生物膜的最佳作用参数：运用荧光分光光度仪检测ALA在生物膜内的吸收和转化为PpIX的情况,研究ALA在白色念珠菌生物膜内的最佳参数,确定最佳ALA孵育时间和最佳ALA用药浓度,为之后的实验提供理论依据。3.评估ALA-PDT对白色念珠菌生物膜的影响：本部分实验建立在前两部分研究的基础上,利用CLSM检测ALA-PDT过程中不同光照剂量对白色念珠菌生物膜活性的抑制作用,计算杀菌率的变化,并定量分析ALA-PDT对生物膜结构的破坏效果,计算生物膜平均厚度的变化,最终确定最佳光照剂量。结果：1. 白色念珠菌生物膜的体外模型建立白色念珠菌ATCC90028培养48h后,经结晶紫染色及光学显微镜观察,可见生物膜紧密附着于载体底部,不被外力吹打和PBS洗涤所破坏。经荧光双染色及CLSM观察,可见典型的生物膜特征：具有一定厚度的主要由细胞外基质组成的紧密膜状结构。XTT减低法分析显示48h的生物膜生长基本饱和,进入了成熟的稳定状态。2.ALA对白色念珠菌生物膜的最佳作用参数白色念珠菌生物膜与ALA孵育后,CLSM下可见PpIX砖红色荧光,说明白色念珠菌生物膜能够吸收ALA并转化为PpIX。通过根据ALA孵育时间和ALA用药浓度的交叉实验发现,荧光强度的变化具有一定的时间依赖性,但不具有浓度依赖性。本实验最终以ALA孵育时间为5h、ALA用药浓度为15mM作为ALA对白色念珠菌生物膜的最佳作用参数。3. ALA-PDT对生物膜的抑制效果经过不同剂量的红光照射后,CLSM双染色观察到死菌数量随光照剂量的增加而增加,生物膜的结构也随光照剂量的增加而疏松,生物膜的平均厚度随光照剂量的增加而减少。当光剂量达300J/cm2时,杀菌率接近80%,生物膜平均厚度缩小超过一半。结论：1.白色念珠菌ATCC90028可以经体外培养48h形成稳定的白色念珠菌生物膜模型。2.白色念珠菌生物膜能够吸收外源性ALA并转化为具有光敏性的物质PpIX。3. ALA-PDT能够对白色念珠菌生物膜内的细胞活性起抑制作用,并且对生物膜结构本身也一定的破坏作用。4. ALA-PDT作用于体外白色念珠菌生物膜的最佳作用参数：ALA最佳孵育时间为5h,ALA最佳用药浓度为15mM, ALA-PDT最佳光照剂量为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..

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R780.2

【共引文献】