曲古菌素A对三唑类药物体外抗白念珠菌活性的增效作用及机制研究

发布时间：2018-03-09 18:53

  本文选题：表观遗传学　切入点：白念珠菌　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：第一部分曲古菌素A对唑类药物体外抗白念珠菌活性增效作用的研究 第一章曲古菌素A对唑类药物体外抗白念珠菌等常见病原真菌活性的影响 目的选择敏感性不同的白念珠菌菌株、组蛋白去乙酰化酶基因缺失株及其它常见病原酵母菌和丝状真菌,研究曲古菌素A对氟康唑、伊曲康唑和伏立康唑抗白念珠菌等常见病原真菌的活性是否有增效作用。方法微量稀释法检测0.25μg/m1曲古菌素A与氟康唑、伊曲康唑和伏立康唑分别合用时对白念珠菌敏感株、耐药株和常见病原真菌的最低抑菌浓度,并检测白念珠菌的最小杀菌浓度,检测组蛋白去乙酰化酶编码基因缺失株HDA1△和RPD3△对三种唑类药物的体外敏感性。结果0.25μg/ml曲古菌素A与氟康唑、伊曲康唑和伏立康唑合用可不同程度地减少白念珠菌的拖尾现象,或表现为生长孔菌量减少,少数菌株的24h最低抑菌浓度降低,对最小杀菌浓度无明显影响；曲古菌素A不影响其它常见病原酵母菌和丝状真菌与唑类药物的体外敏感性；与同源株比较,HDA1△和RPD3△,体外敏感性增加。结论曲古菌素A对唑类抗真菌药物体外抗白念珠菌活性有增效作用,对其体外杀菌作用无影响。曲古菌素A与唑类抗真菌药物合用对除白念珠菌外的常见病原酵母菌和丝状真菌的体外敏感性无增效作用。组蛋白去乙酰化酶编码基因HDA1和RPD3可能与白念珠菌体外敏感性相关。 第二章曲古菌素A对唑类药物体外增效作用的机制初探 目的研究白念珠菌组蛋白去乙酰化酶编码基因的表达水平,以及组蛋白去乙酰化酶编码基因和外排泵基因在体内外获得性耐药形成过程中表达水平的变化。应用罗丹明123(Rh123)检测曲古菌素A对白念珠菌外排泵功能的影响。方法体外诱导白念珠菌对氟康唑耐药,收集药物作用前的白念珠菌和氟康唑环境下的体内外获得性耐药的白念珠菌。Trizol法提取白念珠菌RNA,白念珠菌ACT1作为内参基因,荧光定量RT-PCR检测白念珠菌临床株组蛋白去乙酰化酶编码基因HDA1、RPD3、 Hos1、HOS2、HOS3的表达水平,以及组蛋白去乙酰化酶编码基因和外排泵基因CDR1、CDR2、MDR1和FLU1在体内外获得性耐药形成过程中表达水平的变化。应用荧光染料Rh123处理白念珠菌,荧光分光光度计检测白念珠菌临床株、体外诱导耐药菌株在曲古菌素A作用下的荧光变化,以检验白念珠菌外排泵功能的变化。结果药物作用前,白念珠菌的组蛋白去乙酰化酶编码基因有不同程度表达,与菌株自身敏感性和曲古菌素A对菌株的增效作用无关；体内外获得性耐药形成过程中HDA1、RPD3、HOS1、HOS2和HOS3表达水平有不同程度增高,外排泵基因CDR1、CDR2、MDR1表达水平呈不同程度增加,LU1表达水平变化不明显。曲古菌素A对白念珠菌临床株短时间作用后,外排泵功能无明显变化,氟康唑／曲古菌素A诱导组的Rh123荧光值高于氟康唑诱导组的Rh123荧光值。结论组蛋白去乙酰化修饰可能是白念珠菌外排泵基因水平变化的上游机制之一。 第二部分念珠菌对常见抗真菌药物的体外敏感性测定 第一章微量稀释法检测念珠菌对抗真菌药物敏感性的两种观察结果方法的比较 目的应用微量稀释法检测念珠菌体外抗真菌药物敏感性,比较肉眼观察和体视显微镜观察结果的一致性及优缺点。方法参考CLSI的微量稀释法M27-A3,检测203株念珠菌对4种唑类药物(咪康唑、酮康唑、益康唑、联苯苄唑)及4种非唑类药物(制霉菌素、利拉萘酯、萘替芬、特比萘芬)的体外敏感性,并同时应用肉眼和体视显微镜两种方法观察结果。结果1.唑类药物：咪康唑、酮康唑、益康唑和联苯苄唑的一致性范围为：87.68%～96.06%；2.非唑类药物：制霉菌素、利拉萘酯、萘替芬、特比萘芬的一致性范围为91.63%～99.51%。3.与肉眼观察比较,体视显微镜观察结果具有以下优点：成像有立体感；可以观察到肉眼看不见的微量菌生长；易于区分待测菌和污染菌生长；4.除利拉萘酯、萘替芬和联苯苄唑外,咪康唑、酮康唑等均对念珠菌有良好的体外抑菌作用。结论对于微量稀释法体外检测念珠菌的抗真菌药物敏感性,应用体视显微镜可以辅助观察结果,且与肉眼读取结果有较好一致性,两种方法结合有助于体外抗真菌药物敏感性结果的判断。 第二章卢立康唑及其它6种咪唑类抗真菌药物对常见念珠菌的体外活性检测 目的评价卢立康唑及其它6种咪唑类药物对临床分离常见念珠菌的体外敏感性。方法参考CLSI的微量稀释法M27-A3方案,检测5种共183株临床分离念珠菌对卢立康唑、酮康唑、咪康唑、益康唑、克霉唑、舍他康唑、联苯苄唑7种咪唑类药物的体外敏感性。结果酮康唑、咪康唑、益康唑、克霉唑、舍他康唑和联苯苄唑的体外最低抑菌浓度范围(几何均数)分别为0.03-8(0.067)μg/ml、0.03-16(0.071)μg/ml、0.03-8(0.207)μg/ml、0.03-8(0.061)μg/ml、0.03-16(0.187)μg/ml.和0.03～16(1.050)μg/ml。抗真菌新药卢立康唑对5种念珠菌均有较好的体外敏感性,最低抑菌浓度范围0.03～8μg/ml,几何均数为0.087μg/ml, MIC50和MIC90分别为0.06μg/ml和0.5μg/ml。包括卢立康唑在内,各受试药物均有部分菌株相对不敏感。结论除联苯苄唑外,卢立康唑等其它6种咪唑类药物均对念珠菌有良好的体外抗菌活性,但存在少数菌株相对不敏感。
[Abstract]:Study on the synergistic effect of triacycin A on the activity of anti Candida albicans
The effect of triacycin A on the activity of the common pathogenic fungi of Candida albicans against Candida albicans in the first chapter
Objective to select the different sensitivity of Candida albicans strains, histone deacetylase gene deletion strains and other pathogenic yeasts and filamentous fungi, study of trichostatin A on whether there is a synergistic effect of fluconazole, itraconazole and voriconazole against Candida albicans and other pathogenic fungi. The activity of A and fluconazole detection method of micro dilution method 0.25 g/m1 in archaea, itraconazole and voriconazole were combined to Candida albicans strains, the minimum inhibitory concentration of drug resistant strains and pathogenic fungi, and to detect the minimal bactericidal concentration of Candida albicans, detection of histone acetylation in vitro sensitivity encoding gene deletion strains HDA1 and RPD3 Delta Delta on three kinds of azole drugs. Results in A and fluconazole 0.25 g/ml Qugu bacteria, itraconazole and voriconazole combined reduced smearing of Candida albicans, or for growth of Kong Jun Reduce 24h, the minimum inhibitory concentration of a few strains were decreased and had no obvious effect on the minimum bactericidal concentration; trichostatin A does not affect the sensitivity of other common pathogenic yeasts and filamentous fungi with azole drugs in vitro; and homologous strains, HDA1 and delta RPD3 Delta, in vitro sensitivity increased. Conclusion trichostatin A had synergism effect of anti Candida albicans in vitro activity of azole antifungal drugs had no effect on the in vitro bactericidal action of trichostatin A and azole antifungal drug sensitivity of Candida albicans in the common pathogenic yeasts and filamentous fungi in vitro. No synergistic effect of histone deacetylase gene encoding HDA1 and RPD3 and in vitro susceptibility of Candida albicans.
A preliminary study on the mechanism of the synergistic effect of trazocin A on azolic drugs in the second chapter
Objective to study the expression of Candida albicans histone acetyltransferase encoding gene, expression and histone deacetylase gene encoding and efflux pump gene acquired resistance in vitro and in vivo formation process. The application of Luo Danming 123 (Rh123) to detect effects of trichostatin A on Candida albicans efflux pump function the method for in vitro induction of Candida albicans resistant to fluconazole, extraction of Candida albicans RNA Candida albicans.Trizol was collected before the drug effects of Candida albicans and fluconazole environment in vivo and acquired drug resistance of Candida albicans ACT1 as reference gene, fluorescence quantitative RT-PCR detection of clinical isolates of Candida albicans histone deacetylase gene encoding HDA1, RPD3, Hos1, HOS2, HOS3 expression, and histone deacetylase gene encoding and efflux pump genes CDR1, CDR2, MDR1 and FLU1 during the formation of acquired drug resistance in vitro and in vivo in table The change of the level of application. The fluorescent dye Rh123 treatment of Candida albicans, fluorescence spectrophotometer detection of clinical isolates of Candida albicans resistant strains in vitro, fluorescence changes in trichostatin A under the action of the change of pumping function to test the Candida albicans outside. The results of drug action, Candida albicans histone deacetylase genes encoding enzymes have different levels of expression, has nothing to do with the strain sensitivity and trichostatin A synergistic effect on the strains; in vivo acquired drug resistance during the formation of HDA1, RPD3, HOS1, HOS2 and HOS3 expression levels increased, efflux pump gene CDR1, CDR2, MDR1 expression level increased, the expression level of LU1 did not change significantly. Trichostatin A in Candida albicans clinical isolates after a short period of time, the function of efflux pump has no obvious change, Rh123 fluorescence / fluconazole induced by trichostatin A group was higher than that of fluconazole induced group Rh The 123 fluorescence value. Conclusion histone deacetylation modification may be one of the upstream mechanisms of the change in the gene level of Candida albicans efflux pump.
In vitro sensitivity of second Candida albicans to common antifungal agents
Comparison of two methods for detecting susceptibility to Candida albicans against fungal drugs in the first chapter
Antifungal susceptibility testing of Candida albicans in vitro by microdilution method, the consistency of the advantages and disadvantages of visual observation and stereological microscopy results. Microdilution reference method M27-A3 CLSI, detection of 203 strains of Candida to 4 azole drugs (miconazole, ketoconazole, miconazole, bifonazole) and 4 non azole drugs (nystatin, liranaflate, naftifine, terbinafine) in vitro sensitivity of two methods and application of visual and stereo microscope. Results 1. azole drugs: ketoconazole, miconazole, Econazole and bifonazole consistency range 87.68% ~ 96.06%; 2. non azole drugs: nystatin, liranaflate, naftifine, terbinafine consistency range is 91.63% ~ 99.51%.3. compared with the naked eye, microscope observation has the following advantages: imaging three-dimensional sense can be observed; The naked eye cannot see the growth of trace bacteria; bacteria and bacteria to distinguish pollution to be tested; 4. except liranaftate, butenafine and bifonazole, miconazole, ketoconazole, have good antibacterial effect in vitro of Candida albicans. Conclusion for antifungal drug sensitivity in vitro microdilution detection of Candida albicans the results can be used to observe, auxiliary stereo microscope, and with the naked eye to read the results have better consistency, two methods contribute to the in vitro antifungal susceptibility of the judgment.
In vitro activity detection of Candida common Candida in the second chapter of luopazolazole and other 6 imidazole antifungal agents
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