氟康唑与他克莫司联合抗非白色念珠菌的作用与机制研究

发布时间：2018-04-14 00:23

本文选题：非白色念珠菌 + 药物联合　；参考：《山东大学》2014年硕士论文

【摘要】：背景：近年来,非白色念珠菌由于其较高的临床分离率和对氟康唑具有较高的获得耐药性或固有耐药性,越来越引起人们的关注和重视。在临床常见非白色念珠菌中,光滑念珠菌是第二位人类最常见的病原菌,具有较高的获得耐药性；克柔念珠菌虽然检出率不高,但对氟康唑天然耐药。它们较高的耐药性给临床成功治疗非白色念珠菌感染带来了挑战。综合考虑临床常见非白色念珠菌的检出率和耐药性,本课题选择光滑念珠菌和克柔念珠菌作为研究对象。抗真菌药物品种有限,而开发一种新的抗真菌药物耗时耗资巨大。因此,为应对真菌不断增加的耐药性,越来越多的研究者关注于抗真菌药物和非抗真菌药物的联合治疗。他克莫司经常用于器官移植患者的免疫抑制治疗,而这些病人更容易遭受真菌感染。前期研究表明氟康唑和他克莫司联用对耐药的白色念珠菌有强大的协同作用,二者联用对光滑念珠菌和克柔念珠菌是否也有协同作用值得研究。目的：本研究旨在评价氟康唑和他克莫司联合对不同敏感性的光滑念珠菌和克柔念珠菌的体外静态和动态抗真菌作用,并探索其协同作用可能的机制。方法：分别采用棋盘法和时间-杀菌曲线法研究氟康畔和他克莫司联用对不同敏感性的光滑念珠菌和克柔念珠菌的体外静态和动态抗真菌作用。根据M27-A3抗真菌药物敏感性测定方案,以XTT法测定两药联合作用24h和48h时对不同敏感性分离株的MIC值,以部分抑菌浓度指数(FICI)评价两药联合作用效果；对于动态联合作用评价,分别在药物干预后的0,6,12,24和48h采用菌落计数法进行菌落计数,然后用药物干预时间作横坐标、每毫升菌液菌落数的常用对数值作纵坐标描绘不同药物处理后的时间-杀菌曲线。药物联合抗真菌作用机制研究包括：加入钙通道抑制剂贝尼地平,以平板划线法直观评价抑制钙通道对两药联合抗真菌作用的影响；以实时定量PCR法测定氟康唑耐药基因ERG11, CDR1, PDH1和SNQ2在不同药物干预后的相对表达量；用罗丹明6G作荧光染色剂,以流式细胞术评价他克莫司对真菌泵出氟康唑的影响。结果：他克莫司增加了剂量依赖性敏感(MIC=32μg/mL)以及耐药的光滑念珠菌和全部克柔念珠菌对氟康唑的敏感性,但菌株的敏感性不同,MIC值降低的程度也不同。对于MIC值大于等于256μg/mL的光滑念珠菌和克柔念珠菌菌株,他克莫司的加入使得氟康唑的MIC值降为原来的1/16,FICI值小于0.1；对于MIC值为32μg/mL的光滑念珠菌和克柔念珠菌菌株,他克莫司的加入使氟康唑的MIC值变为原来的1/4,FICI值约等于0.25；然而对于MIC值为8μg/mL的光滑念珠菌菌株,他克莫司的加入并没有降低氟康唑的MIC值。动态抗真菌作用实验结果进一步证明了两药的协同作用：与氟康唑单用组相比,两药联合对光滑念珠菌和克柔念珠菌在24h时每毫升菌液CFU的常用对数值分别下降了2.31和2.25个单位。另外,以平板划线法观察菌株在药物作用后的生长情况,结果表明贝尼地平的加入增加了两药联合抗光滑念珠菌和克柔念珠菌的作用。对光滑念珠菌进一步的机制研究表明氟康唑和他克莫司联用显著降低了ERG11和SNQ2基因的表达水平(表达量分别减少了90%和66%),显著增加了CDRI基因的表达,对PDH1基因的表达无显著影响。流式细胞术的分析结果表明他克莫司抑制了氟康唑的外排,特别是在加入他克莫司作用40分钟后抑制作用最明显(胞内荧光强度是对照组的2.76倍)。结论：氟康唑和他克莫司联用对光滑念珠菌和克柔念珠菌展现出协同作用。抑制钙离子通道能够增强两药协同抗真菌作用,说明两药协同抗真菌作用与钙调节有关；两药协同抗光滑念珠菌的作用机制与减少了ERG11和SNQ2基因的表达和抑制了氟康唑的外排有关。他克莫司和氟康唑联合用药可能是克服光滑念珠菌和克柔念珠菌对氟康唑耐药性的方法,本课题的研究给抗真菌药物的研发提供了思路。
[Abstract]:Background : In recent years , non - Candida albicans has attracted more and more attention due to its high clinical separation rate and its high resistance to drug resistance or inherent drug resistance . In clinical non - candida albicans , Candida glabrata is the most common pathogenic bacterium in the second place , and has high resistance to resistance ;
Although the detection rate of Candida albicans is not high , it presents a challenge to the successful treatment of non - candida albicans infection . It has been considered that the combination therapy of antifungal drugs and non - antifungal drugs has been paid more and more attention to the detection rate and drug resistance of non - candida albicans .

Objective : To evaluate the in vitro static and dynamic antifungal effects of Fluconazole and Fluconazole in vitro static and dynamic antifungal activity against Candida albicans and Candida albicans , and to explore possible mechanisms for its synergistic action .

Methods : In vitro static and dynamic antifungal effects of Fluconazole and Kamox on the static and dynamic antifungal activity of Candida albicans and Candida albicans were studied by checkerboard method and time - sterilization curve method . The MIC values of different sensitive isolates were determined by XTT method at 24 h and 48 h .
For the evaluation of dynamic combined action , the colony counting method was used to count the colony count by colony counting method at 0 , 6 , 12 , 24 and 48 hours after drug intervention , then the time - sterilization curve after the treatment of different drugs was plotted with the drug intervention time as the abscissa and the logarithm of the colony count per milliliter .
Real - time quantitative PCR method was used to determine the relative expression of ER1 , CDR1 , PDH1 and SNQ2 of Fluconazole in different drug interventions .
Rodamin 6G was used as a fluorescent stain to evaluate the effect of fluconazol on fungal pump by flow cytometry .

RESULTS : The sensitivity of the dose - dependent sensitivity ( MIC = 32渭g / mL ) and the resistance of Candida glabrata and Candida albicans to fluconazol were increased . The sensitivity of the strains was different , and the MIC values were decreased . The MIC values of fluconazol were reduced to 1 / 16 and the FICI value was less than 0.1 for the strains with MIC values greater than or equal to 256 渭g / mL .
For Candida and Candida strains with MIC values of 32 渭g / mL , the MIC values of fluconazol were changed to 1 / 4 with a FICI value of approximately 0.25 ;
The results of flow cytometry showed that the combination of fluconazol and Kamox increased the expression level of the two drugs ( 90 % and 2.25 units , respectively ) . The results showed that the combination of fluconazol and Kamox increased the expression level of the two drugs ( 90 % and 66 % , respectively ) .

Conclusion : The combination of Fluconazole and Fluconazole showed a synergistic effect on Candida albicans and Candida albicans . The inhibition of calcium channel could enhance the synergistic antifungal effect of two drugs , suggesting that the synergistic antifungal effect of two drugs was related to calcium regulation .
The action mechanism of the two drugs in combination with anti - smooth candida is related to the reduction of the expression of the ER1 and SNQ2 genes and the inhibition of the efflux of fluconazol . The combination of tamostat and fluconazol can be a method for overcoming the resistance of Candida albicans and Candida albicans to fluconazol , and the research of the subject provides a thought for the development of antifungal drugs .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969

【参考文献】