阿萨希毛孢子菌氟康唑耐药与ERG11基因表达关系的研究

发布时间：2018-04-17 02:13

  本文选题：阿萨希毛孢子菌 + 氟康唑　； 参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景与目的阿萨希毛孢子菌(Tricrosporon asahii,T.asahii)是引起毛孢子菌感染(Trichosporonosis)的主要致病菌。近年来,随着抗菌药物的不规范使用、免疫抑制剂的广泛使用以及艾滋病患者的增加,阿萨希毛孢子菌感染的发病率也越来越高。唑类药物因其抗真菌谱广、不良反应小等优势被广泛用于真菌感染,包括毛孢子菌感染的治疗。但是,近几年报道出了毛孢子菌对唑类药物较高的MIC(Minimum inhibitory concentration)值,甚至是耐药。ERG11基因编码的羊毛甾醇14α-去甲基化酶为唑类抗真菌药物的作用靶点,唑类药物与其特异性结合后抑制该酶活性,从而阻遏真菌细胞膜重要成分——麦角甾醇的合成。既往已有研究显示ERG11基因突变和或表达上调可引起白念珠菌等多种念珠菌以及新型隐球菌等真菌对唑类药物耐药。但尚未有关于ERG11基因表达在阿萨希毛孢子菌唑类耐药中作用的研究报道。故本研究欲通过荧光定量PCR(Real-time PCR=q-PCR)技术检测多对敏感菌株与耐药菌株的ERG11基因的表达情况,探讨ERG11基因在阿萨希毛孢子菌唑类耐药中的作用以及基因表达量与药物浓度的关系。为进一步研究阿萨希毛孢子菌的耐药机制及新药靶点的开发奠定基础。方法通过不断增加氟康唑药物浓度方法体外诱导获得多株阿萨希毛孢子菌耐药菌株,然后再将敏感菌株与耐药菌株同时置于含有0~64μg/ml浓度氟康唑的酵母蛋白胨葡萄糖培养基(YPD)中培养,提取RNA,使用q-PCR方法检测所有受试菌株的ERG11基因的m RNA表达量。比较该基因在氟康唑敏感菌株与耐药菌株中的表达差异,以及在不同浓度氟康唑作用下表达量的变化。并检测分析氟康唑体外诱导过程中各菌株ERG11基因表达的变化。结果共获得6株体外诱导成功的耐药菌株。在无药情况下,阿萨希毛孢子菌耐药株组ERG11基因表达(7.542±5.311)高于敏感株组(1.014±0.012),两组比较t=3.002,p=0.03,差异有统计学意义。在不同浓度氟康唑作用下,耐药株组ERG11m RNA水平分别为9.183±3.226,13.657±5.428,15.292±7.007,13.720±8.550,13.949±2.960,13.123±6.429,亦高于敏感株组3.281±2.068,3.459±1.923,3.242±2.530,3.651±0.728,3.969±1.924,3.824±1.875,均p0.05,有统计学意义。但ERG11表达量与氟康唑浓度之间差异无显著相关性(耐药株rs=0.229,p=0.096;敏感株rs=0.166,p=0.357)。且在诱导耐药过程中,虽然各受试菌株的ERG11表达量随着其对氟康唑的耐药性上升均呈现出上升趋势,但只有2株菌株的耐药性与其ERG11表达量之间有显著等级相关性(BZP902:rs=0.865,p=0.003;BZP705:rs=0.847,p=0.002)。结论ERG11基因的表达水平与阿萨希毛孢子菌氟康唑耐药有关,但ERG11基因过表达是否会导致阿萨希毛孢子菌氟康唑耐药,及其具体机制还有待于进一步研究。
[Abstract]:Background & objective Tricrosporon asahiiae (T. asahii) is the main pathogen of Trichosporonosism.In recent years, with the irregular use of antimicrobial agents, the widespread use of immunosuppressants and the increase of AIDS patients, the incidence of Asaxia sporocystis infection is also increasing.Azoles are widely used in the treatment of fungal infections, including Trichosporum, due to their wide antifungal spectrum and small adverse reactions.However, in recent years, it has been reported that the higher value of MIC(Minimum inhibitory concentration of Cryosporium to azoles, and even the target of wool sterol 14 伪 -demethylase encoded by the resistance. ERG11 gene, as the target of antifungal agents of azoles, has been reported in recent years.The activity of the enzyme was inhibited by the specific binding of zolium, which inhibited the synthesis of ergosterol, an important component of fungal cell membrane.Previous studies have shown that mutation and up-regulation of ERG11 gene can induce resistance of Candida albicans and Cryptococcus neoformans.However, there have not been any studies on the role of ERG11 gene expression in the resistance of Asaxia sporidium to azotrazole.Therefore, the purpose of this study was to detect the expression of ERG11 gene in sensitive and resistant strains by fluorescence quantitative PCR(Real-time PCR q-PCR technique, and to explore the role of ERG11 gene in the resistance of Asaxia sporidium and the relationship between gene expression and drug concentration.It will lay a foundation for further study on the drug resistance mechanism of Asaxia and the development of new drug targets.Methods by increasing fluconazole concentration in vitro, many strains of Asaxia sporidium resistant strains were obtained.Then, the sensitive strains and the drug-resistant strains were cultured in yeast peptone glucose medium containing fluconazole (0 渭 g/ml) at the same time. The q-PCR method was used to detect the m RNA expression of ERG11 gene in all the tested strains.To compare the difference of expression of this gene between fluconazole-sensitive strain and drug-resistant strain, and the change of expression quantity under different concentration of fluconazole.The changes of ERG11 gene expression during fluconazole induction in vitro were detected and analyzed.Results A total of 6 drug-resistant strains were successfully induced in vitro.In the absence of drug, the expression of ERG11 gene was 7.542 卤5.311 in the resistant strains of Asaxia and 1.014 卤0.012 in the susceptible strains. The difference between the two groups was statistically significant.The levels of ERG11m RNA in the resistant strain group were 9.183 卤3.226, 13.657 卤5.428, 15.292 卤7.007, 13.720 卤8.550, 13.949 卤2.960,949 卤6.429, and 3.281 卤2.0683.459 卤1.9233.242 卤2.530, 3.651 卤0.728, 3.969 卤1.9243.824 卤1.875, respectively.However, there was no significant correlation between the expression of ERG11 and fluconazole concentration (0.229p0. 096 in resistant strain, 0.166 in susceptible strain and 0.357 in susceptible strain).In the course of inducing drug resistance, although the ERG11 expression of each tested strain showed an upward trend with the increase of fluconazole resistance, only two strains showed a significant grade correlation with their ERG11 expression, BZP902: Rs902: Rs902: Rs0.003 BZP705: rs0.847 p0.002.Conclusion the expression level of ERG11 gene is related to fluconazole resistance of Asaxia sporidium, but whether the overexpression of ERG11 gene leads to fluconazole resistance of Asaxia sporidium remains to be further studied.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R756

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