盐酸氨溴索与氟康唑联合对抗耐药白色念珠菌的作用及机制研究

发布时间：2018-01-19 09:49

本文关键词： 盐酸氨溴索氟康唑耐药白色念珠菌协同作用协同机制　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景近年来,真菌,尤其是侵袭性真菌,已经成为引起院内感染的重要病原菌。最常见的侵袭性真菌主要包括念珠菌和曲霉,其中念珠菌中的白色念珠菌是最为常见的人体机会致病菌之一,由白色念珠菌造成的感染致死率也很高,已超过30%,这主要是由于目前临床上可用的抗真菌药物异常匮乏以及该病原菌易产生高耐药性所致。目前,临床可用的抗真菌药物种类不多,仅有唑类、棘白菌素类、多烯类、嘧啶类和烯胺类等为数不多的几个品种。在这些抗真菌药物中,作为唑类抗真菌药物之一的氟康唑由于其具有生物利用度高和毒性低的特点,已成为临床上治疗白色念珠菌感染的首选药物。然而,广泛、高频率、高剂量使用氟康唑却导致了白色念珠菌耐药性的出现,使得临床上对抗耐药白色念珠菌所致的感染变得十分棘手。此外,白色念珠菌还容易在体内留置导管上形成难以清除的生物膜,而生物膜的形成也在一定程度上增加了其耐药性。因此,白色念珠菌的耐药现象以及耐药白色念珠菌所致感染为临床治疗带来的挑战已经不容忽视。目前尚未找到克服白色念珠菌耐药现象的有效途径和手段,所以感染耐药白色念珠菌的医院患者只能接受高毒性或昂贵的抗真菌药物的治疗。因此,开发能够克服真菌耐药的治疗手段迫在眉睫。目前,寻找可以增加氟康唑敏感性的非抗真菌药物,将其和氟康唑联合用药来对抗耐药白色念珠,已成为较为成功的方法,备受关注。盐酸氨溴索具有强大的祛痰作用,能改善呼吸作用,且毒性十分低,因而被广泛用于呼吸系统疾病的治疗。到目前为止,该药在临床上的疗效、耐受性和安全性均已被证实和肯定。近来实验研究表明,盐酸氨溴索单用还具有抗微生物作用,可以对抗敏感白色念珠菌、非白色念珠菌和某些细菌。然而,目前尚无关于盐酸氨溴索与氟康唑联用抗真菌的相关实验研究报道。目的本实验的目的首先是测定氟康唑与盐酸氨溴索的体内、外抗念珠菌活性,分析二者联用是否有协同抗真菌作用,并进一步探索氟康唑与盐酸氨溴索的药物组合对耐药白色念珠菌的协同抗真菌作用机制。方法按照CLSI M27-A3药物敏感性测定方案,采用微量稀释法测定氟康唑和盐酸氨溴索单用或联用分别对敏感白色念珠菌、耐药白色念珠菌的体外抗真菌作用,并以部分抑菌浓度指数(fractional inhibitory concentration index,FICI)模型和生长差异百分率(percentage ofgrowth difference,ΔE)模型共同地评价这两个药物联合抗真菌作用效果。使用大蜡螟作为白色念珠菌感染模型,通过测定大蜡螟的生存率、载菌量和组织学改变综合评价氟康唑和盐酸氨溴索单用与联用在体内的联合作用效果。在机制研究方面,使用罗丹明6G作为氟康唑的荧光替代物,借助流式细胞仪评价盐酸氨溴索对耐药白色念珠菌药物外转运泵活性的影响。此外,本研究还测定了药物联用前后对耐药白色念珠菌的菌丝生长情况和胞外磷脂酶活性的变化,评价该药物组合对耐药白色念珠菌毒力因子表达情况的影响,并以实时定量PCR法测定药物联用对菌丝相关基因(HWP1)和磷脂酶编码基因(PLB1和PLC1)的影响。结果1药物联用的体外抗真菌作用加入盐酸氨溴索(浓度为128μg/mL)可以使氟康唑对耐药白色念珠菌CA10和CA16的MIC值从512μg/mL以上降至2μg/mL,FICI模型和ΔE模型均表示两药在体外联用具有很好的协同抗耐药白色念珠菌作用。两药在体外联用对抗敏感白色念珠菌作用的FICI值均大于1、ΔE绝对值均远大于200%,表明该药物组合对敏感白色念珠菌没有协同作用。2药物联用的体内抗真菌作用氟康唑和盐酸氨溴索单用时对提高感染耐药白色念珠菌的大蜡螟的生存率没有显著的作用,但是联用组可以明显提高感染后大蜡螟的生存率,由此初步确定了该药物组合的体内抗真菌疗效。另外,与其他组三相比,两药联用组可明显降低感染大蜡螟体内的载菌量。通过观察四组感染耐药白色念珠菌后的大蜡螟病理组织切片之后我们发现,与其他组三相比,药物联用组的大蜡螟体内黑色真菌斑块明显减少。我们的结果证实了氟康唑联合盐酸氨溴索在体内的抗耐药白色念珠菌作用。3盐酸氨溴索可显著抑制药物转运泵的活性罗丹明6G和氟康唑,两者都是真菌细胞膜上药物转运泵的作用底物。本实验将罗丹明6G用作氟康唑的替代物。与对照组相比,盐酸氨溴索干预后,白色念珠菌胞内罗丹明6G明显吸收加快、外排明显减慢。由此可见,两药联用的协同抗真菌机制可能与它们抑制真菌细胞膜上的药物转运泵的活性进而增加氟康唑的胞内浓度有关。4盐酸氨溴索联合应用氟康唑可抑制菌白色念珠菌毒力因子的表达4.1药物联用可抑制菌丝生长借助荧光显微镜可以看到,对照组、氟康唑单用组和盐酸氨溴索单用组均有出大量含有细长的菌丝,且菌丝有聚集成团现象,但是在药物联用组中的菌丝十分稀少,且菌丝很短,甚至有被破坏的现象。此外,与对照组相比,药物联用组中的菌丝生长调控基因(HWP1)明显降低。由此我们可以确定,该药物组合联用的协同抗真菌机制可能与抑制耐药白色念珠菌的菌丝的生长有关。4.2药物联用可抑制胞外磷脂酶活性白色念珠菌可分泌胞外磷脂酶,卵黄琼脂培养基是一种能够快速检测该酶活性的培养基。结果发现,药物单用组的Pz值与对照组相比无显著性差异,但是药物联用组的Pz值对照组相比却明显升高,表明盐酸氨溴索与氟康唑联合应用具有很强的抑制耐药白色念珠菌胞外磷脂酶活性的作用。通过进一步的实时定量PCR的结果可以发现,药物联用可明显降低磷脂酶编码基因PLB1和PLC1的表达,进一步揭示了该药物组合对耐药白色念珠菌胞外磷脂酶活性具有抑制作用,同时也表明PLB1和PLC1可能为该药物组合协同抗耐药白色念珠菌作用的潜在靶点。结论本研究揭示了盐酸氨溴索与氟康唑联用对抗耐药白色念珠菌的体内、外作用效果。此外,我们发现该药物组合协同抗耐药白色念珠菌的作用机制与抑制抗真菌药物转运泵活性、抑制毒力因子(菌丝生长、胞外磷脂酶活性)的表达有关。我们的实验第一次揭示了盐酸氨溴索可与氟康唑联用逆转白色念珠菌的耐药性。此外,我们的研究还将有助于新的抗真菌药物靶点的发现。
[Abstract]:Background: in recent years, fungi, especially invasive fungi, has become an important pathogen causing nosocomial infection. The most common invasive fungi including Candida and Aspergillus, Candida in Candida albicans is one of the most common opportunistic human pathogen, caused by Candida albicans infection mortality rate is very high and has more than 30%, which is mainly due to the current clinical available antifungal drugs and the lack of abnormal pathogenic bacteria to produce high resistance caused. At present, the anti fungal drug clinical types are not many, only azole, echinocandins, polyenes, pyrimidine and enamine for several the number of varieties. In these antifungal agents, as one of the azole antifungal drugs fluconazole because of its high bioavailability and low toxicity characteristics, has become the first choice drugs for treatment of Candida albicans infection in clinic. However, extensive, high frequency, high dose of fluconazole has led to the emergence of drug resistance of Candida albicans, the clinical resistant Candida albicans infection becomes very difficult. In addition, Candida albicans also easy in vivo biofilm formation and removal of indwelling catheter, and biofilm formation also has increased to a certain extent the drug resistance. Therefore, drug resistance of Candida albicans and Candida albicans infection caused by drug resistance for clinical treatment of the challenge has been ignored. To overcome the drug resistance of Candida albicans has yet to find effective ways and means of the phenomenon, so the resistance of Candida albicans infection in hospital patients can receive treatment of highly toxic or expensive antifungal drugs. Therefore, to overcome the development of fungal resistance treatment is imminent. At present, looking for non really can increase the sensitivity of fluconazole resistance The bacteria drug, and fluconazole combination against drug-resistant Candida albicans, has become a successful method of concern. Ambroxol hydrochloride has a strong expectorant effect, can improve the respiratory function, and the toxicity is very low, so it is widely used for the treatment of respiratory diseases. So far, the effect of the drug in the clinic the tolerance and safety have been confirmed and affirmed. Recent experimental research shows that ambroxol hydrochloride alone has antimicrobial effects against sensitive Candida albicans, Candida albicans and some bacteria. However, there is no about ambroxol hydrochloride and fluconazole combined with related research reports. The purpose of the anti fungi the first is the determination of fluconazole and ambroxol hydrochloride in vivo, and anti Candida activity, combined with analysis of two have synergistic antifungal effects, and further explore and fluconazole Synergistic antifungal mechanism of ambroxol hydrochloride combination on drug resistance of Candida albicans. Methods according to the CLSI M27-A3 determination method of drug sensitivity determination of fluconazole and ambroxol hydrochloride alone or in combination were sensitive to Candida albicans by microdilution method, the antifungal activity of resistant Candida albicans in vitro, and the fractional inhibitory concentration index (fractional inhibitory concentration index, FICI) model and the growth difference percentage (percentage ofgrowth difference, E) model to evaluate the two common drugs combined with anti fungal effect. The use of wax moth as Candida albicans infection model, the survival rate of determination of wax moth, changes of the comprehensive evaluation of fluconazole and ambroxol hydrochloride alone and their combination with the combined effects of the in vivo effects of microbial load and organization. In terms of mechanism research, using Luo Danming 6G as fluorescence of fluconazole Substitute, with effect evaluation of ambroxol hydrochloride pump on the activity of drug resistant Candida albicans. Flow cytometry. In addition, this study also measured the changes of drug resistance of Candida albicans after mycelial growth and extracellular phospholipase activity, the evaluation of effect of the drug combination on the expression of drug resistance of Candida albicans virulence factor, and real time quantitative PCR method for the determination of drugs on the mycelial associated gene (HWP1) encoding gene and phospholipase (PLB1 and PLC1). The influence of the antifungal effect of the 1 drugs in vitro with ambroxol hydrochloride (concentration 128 g/mL) can make the fluconazole resistant Candida albicans CA10 and CA16 the MIC value from 512 Mu g/mL to 2 mu g/mL, FICI model and E model are delta two drugs have synergistic anti drug resistant Candida albicans in vitro with good medicine against white. Two sensitive in vitro with The role of Candida albicans FICI values are greater than 1, the absolute value of E was greater than 200%, indicating that the drug combination has no synergistic effect of.2 in combination with in vivo antifungal effects of fluconazole sensitive Candida albicans and ambroxol alone had no significant effect on improving the survival rate of Galleria mellonella infection resistant Candida albicans. But the combination group can significantly improve the survival rate of infection after the wax moth, to determine the initial antifungal efficacy of the combination of drugs in vivo. In addition, with other groups of three phase ratio, the two drug combination group can significantly reduce the microbial load of infection of Galleria mellonella in vivo. After infection were observed in four groups of drug resistance of Candida albicans after the wax moth pathological sections we found that with the other groups compared to three drug group the wax moth in black fungus plaque decreased. Our results confirmed that fluconazole combined with ambroxol hydrochloride in vitro Resistance of Candida albicans.3 in Ambroxol Hydrochloride can significantly inhibit drug transporter activity of Luo Danming 6G and fluconazole, both substrates on fungal cell membrane drug transport pump. This experiment will be used as a substitute for Luo Danming 6G of fluconazole. Compared with the control group, the ambroxol hydrochloride intervention, Candida albicans intracellular Luo Danming 6G significantly accelerates absorption, efflux was reduced. Thus, can inhibit the mycelial growth by fluorescence microscope can see that the combination of the two drug combination synergistic antifungal mechanism may be related to their inhibition of drug transport pump on fungal cell membrane and increase the activity of the intracellular concentration of fluconazole combined with ambroxol hydrochloride on.4 fluconazole can inhibit bacteria the expression of virulence factors of Candida albicans 4.1 drug control group, fluconazole monotherapy group and ambroxol alone group had a slender hyphae containing a large number of, And the hyphae are gathered into a group phenomenon, but in combination with hyphae in the group is very scarce, and the mycelium is very short, even the destruction of the phenomenon. In addition, compared with the control group, drug group in the mycelial growth controlling gene (HWP1) was significantly reduced. In this way we can determine, can inhibit the extracellular the phospholipase activity of Candida albicans secreted extracellular phospholipase associated with the drug combination for hypha synergistic antifungal mechanism may be related to the inhibition of drug resistance of Candida albicans growth.4.2 drugs, egg yolk agar is a culture medium for rapid detection of the enzyme activity. The results showed that with the single drug group compared with the Pz value the control group had no significant difference, but the drug group Pz values compared to the control group, but significantly higher, showed a combination of ambroxol hydrochloride and fluconazole has strong inhibition of drug resistance of Candida albicans extracellular phospholipase activity The role of the real-time PCR further results can be found, can reduce the expression of phospholipase PLB1 gene encoding and PLC1 drug combination, further reveals the combination of drug resistant Candida albicans extracellular phospholipase activity has inhibitory effect, also showed the potential targets of PLB1 and PLC1 may have synergistic anti resistant Candida albicans for the drug combinations. Conclusions ambroxol hydrochloride combined with fluconazole resistant Candida albicans in vivo, external effect. In addition, we found that the mechanism of the drug combination synergistic anti Candida albicans resistance and inhibition of antifungal drug transporter activity, inhibition of virulence factors (mycelial growth, extracellular phospholipase activity) the expression. Our study reveals for the first time with fluconazole combined with ambroxol can reverse the drug resistance of Candida albicans. In addition, I have The study will also contribute to the discovery of new antifungal targets.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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