第二军医大学
硕士学位论文
活性氧相关分子对唑类药物药效的影响
姓名：***
申请学位级别：硕士
专业：药理学
指导教师：***
2011-05
活性氧相关分子对唑类药物药效的影响
摘  要
白假丝酵母菌（Candida albicans）是临床最常见的条件致病性真菌，它寄居在人体不同的解剖部位，包
括皮肤、口腔、胃肠道、阴道等，约占所有医院真菌感染的50－60%，能够引起多种疾病，尤其是皮肤感染、粘膜感染以及侵入性深部真菌感染。在正常宿主中，念珠菌是一种无害的共生物，检出率几乎达50%。正常机体有足够的免疫力阻止白假丝酵母菌感染，但是当外界因素打破菌平衡，白假丝酵母菌在局部大量生长繁殖，就会导致感染。随着氟康唑长期大量应用导致真菌耐药性普遍产生，成为临床上失败的主要原因。目前抗真菌药物的联合应用已经成为临床抗真菌的发展方向之一。但国内外报道的联合用药多局限于少数抗真菌药物之间的合用，有些抗真菌药物合用反而产生拮抗作用。活性氧簇（Reactive Oxygen Species，ROS）是细胞有氧代谢过程中产生的一组带有未成对电子的化学性质活泼的含氧自由基，主要包括超氧阴离子、羟基自由基、单线态氧和过氧化氢等。许多国内外文献认为白假丝酵母菌耐药性与活性氧相关，但是直接针对体外应用过氧化氢对唑类药物药效影响的报道较少。
本课题首先通过体外实验棋盘式微量稀释法检测了过氧化氢与氟康唑、伊曲康唑、咪康唑、酮康唑、硫康唑合用对8株临床耐药白假丝酵母菌的MIC80值，计算其相应的FIC指数。结果发现以MIC80值为基础，两药合用对全部8株菌（100％）皆表现出协同作用（FICI<0.5）。不同菌株的协同指数不同，但是均表现出协同效果。其次通过平板纸片扩散试验看到，在加FLC平板上的含过氧化氢药物纸片的抑菌圈与空白平板有显著差异。在各类唑类药物的琼脂平板纸片扩散试验均得到了阳性结果。此外，我们运用另一升高活性氧的药物寡霉素进行纸片扩散实验，结果提示：寡霉素与各类唑类药物也具有协同效果。当使用抗氧化剂与唑类药物合用时，分别通过体外实验棋盘式微量稀释法测定了NAC、GSH和唑类药物
reactive是什么药对8株耐药菌株，5株敏感菌株，酿酒酵母菌株(DY150)，以及SC5314的药物作用，结果显示合用抗氧化剂时，唑类药物的MIC80值会显著提升(8倍以上)。进一步验证了增加ROS会加强唑类的药效，而降低ROS则会减弱唑类药物的药效。同样通过测定杀菌-时间曲线在药物作用24h后，两药合用组的活菌浓度比氟康唑单用组有明显降低，进一步证实了降低ROS则会减弱唑类药物的药效。第三部分验证了敲除ROS相关基因—硫氧化蛋白还原酶基因TRR1后，与野生酵母菌相比，氟康唑、咪康唑的MIC80值减低，结果显示敲除TRR1基因后，菌株对部分唑类药物的敏感性增强，同时我们还测试了敲除trr1基因后的ROS水平验证其对ROS的影响，进一步验证其唑类药物药效的影响。
经本课题的研究，我们发现过氧化氢有较强的协同氟康唑抗耐药白假丝酵母菌作用，合用H2O2对MCZ的影响最大，菌株655、379、502、782、876的FICI值均小
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第二军医大学硕士学位论文
于同组其他药物的FICI值。菌株103，958，649则是FLC与H2O2合用的FICI值最小，同时使用抗氧化剂会减弱唑类药物的药效，而敲除清除ROS基因也会S使菌株对于唑类药物敏感，从多个方面验证了ROS相关分子对于唑类药物的影响，该研究为抗耐药白假丝酵母菌和寻新的抗耐药白假丝酵母菌药物提供了新思路。
关键词：活性氧，唑类药物，药效，H2O2，寡霉素，NAC，GSH
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活性氧相关分子对唑类药物药效的影响
Abstract
Candida albicans (Candida albicans) is the most common clinical conditions of pathogenic fungi, it is housed in the different anatomical parts of the body, including skin, oral cavity, gastrointestinal tract, vagina, accounts for approximately 50-60% of nosocomial fungal infections. It can cause a variety of diseases, especially skin infections, mucosal infections, and invasive fungal infection. In normal hosts, Candida is a common harmless creatures, the detection rate of almost 50%. Normal immune system enough to prevent the body of Candida albicans infections, but when external factors to break the flora balance, a large number of Candida albicans growth and reproduction in the local, it will lead to infection. Along with fluconazole long term lead to fungal resistance to a large number of applications in general has become the main reason of the failure of clinical treatment. The combined application of the current antifungal agents’ antifungal therapy has become a clinical one of the development. However, combination therapy reported more domestic limited to a small number of combination betwe
en antifungal agents, but produce some combination antifungal antagonism, the mechanism of azole drugs and reactive oxygen species (ROS)-related, and medicines for the ROS to the azole Reported less effective. ROS (Reactive Oxygen Species, ROS) are generated during aerobic metabolism of cells in a group with unpaired electron chemically active oxygen free radicals, including superoxide anion, hydroxyl radical, singlet oxygen and And hydrogen peroxide. Many domestic and foreign literature that drug resistance of Candida albicans associated with reactive oxygen species, but applied directly to in vitro hydrogen peroxide on the efficacy of azole drug reported less.
We first to detect hydrogen peroxide combination with fluconazole, miconazole, itraconazole, ketoconazole sulfur on the 8 Uniconazole resistant Candida albicans clinical MIC80 values in vitro checkerboard microdilution method and calculate the corresponding FIC index. The results showed that the MIC80 value is based on two-drug combination of all 8 strains (100%) showed synergy (FICI <0.5). Different strains showed different synergy indexs but both had synergistic effect. Secondy, from the flat disk diffusion test, hydrogen peroxide-containing antibacterial drugs ring in plus FLC plates and a blank plate shows a significant difference. Azole drugs in various types of agar plate disk diffusion test have been positive results. When validate other drugs (oligomycin) which can be increased ROS, the experimental results are as the direct use of hydrogen peroxide, also show significant synergistic effect.When using anti-oxidants combination azole drugs with the
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第二军医大学硕士学位论文
NAC, GSH, 8 azole drug resistant strains, 5 suSC5314eptible strains, yeast strains (DY150), and SC5314, results showed that when combined with antioxidants, MIC80 values of azole drugs will significantly increase (8 times) with checkerboard microdilution method. Verify the efficacy of the reactive oxygen species of azole drugs. By measuring the same sterilization - time curves after 24h in the drug, the two combination therapy group was significantly lower than the fluconazole concentration of live bacteria alone group, confirmed the synergistic effect of the two drugs.The third part we validated the genes trr1 about the ROS knocked out, compared with the wild yeast by checkerboard microdilution assay of fluconazole, miconazole and sulfur MIC80 values have redused, so that,we can see that without TRR1, the yeast became more sensitive to the conazoles. We also tested the level of ROS of knockout gene trr1 to verify effects on ROS, and validate the efficacy of azole drugs, the results are displayed as positive.
The research of this subject, we found that hydrogen peroxide has a strong synergistic effect of fluconazole against Candida albicans drug resistance, the intensity is with a dose dependentant, Tth i
mpact of the MCZ combined with H2O2 is greatest, the FICI of strain 655, 379, 502, 782, 876 were less than the same group of other drugs’ FICI value. The minimum FICI value of strain 103, 958, 649 H2O2 is combined with the FLC, while the use of anti-oxidants will diminish the efficacy of azole drugs, and removal ROS gene strains can also cause resistantant of azole drugs, Validate the the impact of ROS-related molecules for azole drugs from multiple ways, the study provides a new idea for drug resistance in Candida albicans and the search for new drugs against resistant Candida albicans.
Key words: ROS, azole, effective, H2O2, oligomycin, NAC, GSH
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