氨基酸对抗真菌药物的影响及其机制研究

发布时间：2018-08-06 21:18

【摘要】：白念珠菌所致的真菌感染发病率急剧上升,临床上常用的提高真菌感染的治疗效果包括：增大剂量,改变药物类型,两种药物或多种药物组合。两种药物联合使用有很多优势,如降低毒性较大的药物的剂量,提高药物治疗的效果,增加抗真菌谱,延缓真菌耐药性的发生等等。氨基酸作为临床目前非常活跃的治疗药物,具有非常重要的作用,如盐酸赖氨酸氯化钠注射液用于脑供血不足、乙酰半胱氨酸用于治疗慢性支气管炎、组氨酸-维生素u-蛋氨酸用于治疗溃疡病等等。因此,氨基酸很有可能作为抗真菌药物增效剂的来源之一,我们以赖氨酸、半胱氨酸为研究对象,进一步深入研究了赖氨酸、半胱氨酸增强两性霉素B对白念珠菌的作用及可能的机制。本课题主要通过氨基酸与抗真菌药物的相互作用研究,以寻找治疗真菌感染的一种安全、有效的联合用药方案。本课题中我们考察外源性氨基酸对抗真菌药物的影响的研究表明,半胱氨酸自身能显著抑制白念珠菌的生长,不同的氨基酸与不同的抗真菌药物联合使用在体外对白念珠菌的生长具有不同的影响。①氨基酸与卡泊芬净(caspofungin, CAS)合用的相互作用结果：半胱、赖、苏、异亮、精、蛋、丝、亮、丙、甘、苯丙、酪、脯氨酸能显著增强CAS对白念珠菌的作用,天冬氨酸能拮抗CAS对白念珠菌的作用；②氨基酸与咪康唑(miconazole, MCZ)合用的相互作用结果：半胱氨酸能显著增强MCZ对白念珠菌的作用,丝氨酸、甲硫氨酸能拮抗MCZ对白念珠菌的作用；③氨基酸与两性霉素B (amphotericin B, AmB)合用的相互作用结果：半胱、赖、苏、异亮、精、蛋、亮、组、脯氨酸能显著增强A『mB对白念珠菌的作用,鸟氨酸、酪氨酸能拮抗AmmB对白念珠菌的作用；④氨基酸与5-氟胞嘧啶(5-Flucytosine,5-Fu)合用的相互作用结果：半胱氨酸、天冬氨酸、谷氨酸能显著增强5-Fu对白念珠菌的作用,色氨酸、缬氨酸能拮抗5-Fu对白念珠菌的作用；⑤氨基酸与特比萘芬(terbinafine, TER)合用的相互作用结果：半胱、苏、精氨酸能显著增强TER对白念珠菌的作用,甲硫、天冬、谷、苯丙、丙、色氨酸能拮抗TER对白念珠菌的作用；⑥氨基酸与紫草素(shikonin, SK)合用的相互作用结果：半胱、赖、苏、异亮、精、蛋、苯丙、色、鸟、甘、丝、丙氨酸能显著增强SK对白念珠菌的作用,天冬氨酸、亮氨酸能拮抗SK对白念珠菌的作用。首先通过微量液基稀释法检测了20种不同氨基酸对白念珠菌生长的影响以及与不同抗真菌药物相互作用对白念珠菌生长的影响。结果显示,半胱氨酸能显著抑制白念珠菌的生长,苏氨酸对白念珠菌的生长抑制作用较弱,其他氨基酸对白念珠菌的生长无抑制作用,不同的氨基酸与不同的抗真菌药物作用对白念珠菌的生长影响不同。第二部分,考察赖氨酸、半胱氨酸单用以及与AmB合用对白念珠菌和其他菌株的作用,通过spot assay、生长曲线、生存率实验进一步考察了赖氨酸对AmB的抗真菌增效作用以及半胱氨酸的抗真菌作用。实验选取白念珠菌SC5314为研究对象,采用微量液基稀释法测得0.5mM赖氨酸就能增强0.25μg/ml的抗白念珠菌作用；0.25mM半胱氨酸能抑制白念珠菌的生长,也能增强0.25μg/ml AmB的抗白念珠菌作用。同样地,1mM赖氨酸就能增强0.5μg/ml AmB对近平滑念珠菌(0401380,392,90018,22090,22019,0201309),新型隐球菌0201309的抗真菌作用；0.25mM半胱氨酸就能影响克柔念珠菌ACCT2159,光滑念珠菌ACCT28226,近平滑念珠菌22090的生长。Spot aasay实验结果显示,赖氨酸组与空白组敏感性无差异,8mM赖氨酸以上浓度与0.25μg/ml AmB合用能抑制白念珠菌的生长,1mM半胱氨酸就能抑制白念珠菌的生长。生长曲线结果也表明,1mM赖氨酸与0.25μg/ml AmB合用与0.25μg/ml AmB单用相比能较强地抑制白念珠菌的生长。我们也通过XTT还原法考察了AmB与赖氨酸联用对白念珠菌生物被膜形成的影响,结果显示0.5μg/ml AmB与4mM赖氨酸联用对白念珠菌生物被膜的形成以及生物被膜细胞代谢活性的抑制作用较强。同时,菌丝生长实验证实赖氨酸与AmB联用对白念珠菌生物被膜的影响可能是由于对菌丝形成的抑制作用。第三部分,赖氨酸与AmB联用的增效作用机制研究,主要方法有,透射电镜观察两药合用对白念珠菌超微结构的影响,流式细胞仪测定细胞周期观察两药合用对细胞周期的影响,用多功能微板检测仪检测两药合用细胞内活性氧(ROS)的产生量,JC-1试剂盒测定两药合用后细胞内线粒体膜电位水平等实验考察AmB与赖氨酸合用对白念珠菌的氧化损伤作用。实验结果表明,AmB能够升高白念珠菌的内源性活性氧水平,而AmB与赖氨酸合用白念珠菌细胞内活性氧水平更高,赖氨酸组与空白组无明显差异；同时,我们也应用Real-time RT-PCR考察了氧化还原相关基因的表达,结果显示：与AmB单用组相比,AmB与赖氨酸合用ROS清除相关基因Sod2表达水平下降,进一步促进白念珠菌细胞内源性ROS的堆积：然而,与AmB单用组相比,AmB与赖氨酸合用氧化应激相关基因(TRR1、CaMCA1、Cap1、GRP2)表达水平都有不同程度的上调,白念珠菌氧化应激能力提高；另外,AmB能够降低白念珠菌线粒体膜电位水平赖氨酸,而AmB与赖氨酸合用白念珠菌线粒体膜电位水平更低,赖氨酸组与空白组无明显差异；与AmB单用组相比,赖氨酸与AmB合用组细胞分裂过程中的隔膜形成不明显,甚至消失,细胞周期阻滞于G2/M期,使细胞有丝分裂受阻,单用组与空白组无明显差异。第四部分,体内研究结果表明,与体外效果不同,半胱氨酸单用对系统性白念珠菌感染的小鼠没有治疗效果,但是半胱氨酸合用两性霉素B治疗效果优于单用两性霉素B治疗效果,与体外效果一致。综上所述,本课题研究发现,多种氨基酸与抗真菌药物具有体外增效作用；半胱氨酸具有较强的抗真菌活性,在体内外都能增强AmB对白念珠菌的抗真菌活性；赖氨酸在体外能增强白念珠菌对AmB的敏感性,其主要作用机制包括阻止细胞分裂过程中隔膜的形成,阻滞细胞周期中的G2/M期,升高细胞内活性氧,降低线粒体膜电位等；另外赖氨酸与AmB合用还能引起一些氧化还原相关基因的表达发生改变。
[Abstract]:The incidence of fungal infection caused by Candida albicans has risen sharply. The clinical effects of improving fungal infection include increasing dose, changing drug types, two drugs or combination of various drugs. The combination of two drugs has many advantages, such as reducing the dose of toxic drugs, improving the effect of drug treatment, and increasing resistance. Fungal spectrum, retarding the occurrence of fungal resistance and so on. Amino acids are very active in clinical treatment, such as the use of Lysine Hydrochloride and Sodiun Chloride Injection for cerebral blood supply deficiency, acetylcysteine used in the treatment of chronic bronchitis, and the use of vitamin u- methionine in the treatment of ulcerative diseases, and so on. Therefore, amino acids are likely to be one of the sources of antifungal agents. We use lysine and cysteine as the research object to further study the effect and possible mechanism of lysine and cysteine enhanced amphotericin B on Candida albicans. This topic mainly through the interaction of amino acids and antifungal agents. Objective: to find a safe and effective combination regimen for treating fungal infections.
In this study, we examined the effects of exogenous amino acids on the antifungal agents, which showed that cysteine could significantly inhibit the growth of Candida albicans. The combined use of different amino acids and different antifungal agents had different effects on the growth of Candida albicans in vitro. (1) amino acids and caspofungin (CAS). The results of the combined interaction: cysteine, lyophile, shiny, semen, egg, egg, silk, bright, Gump, Gump, phenylpropyl, cheese, proline can significantly enhance the effect of CAS on Candida albicans, and aspartic acid can antagonize the effect of CAS on Candida albicans; and the results of the interaction between amino acids and miconazole (miconazole, MCZ): cysteine can significantly enhance the MCZ against white The effect of Candida, serine and methionine can antagonize the effect of MCZ on Candida albicans; (3) the interaction results of amino acids and amphotericin B (amphotericin B, AmB): cysteine, Lai, Su, shiny, sperm, eggs, bright, group, and proline can significantly enhance the effect of A "mB on Candida albicans, ornithine, tyrosine can antagonize AmmB against Candida albicans." The interaction between the amino acid and 5- fluorocytosine (5-Flucytosine, 5-Fu): cysteine, aspartic acid, and glutamic acid can significantly enhance the effect of 5-Fu on Candida albicans. Tryptophan and valine can antagonize the effect of 5-Fu on Candida albicans; 5. The interaction of amino acid and terbinafine, TER. Results: cysteine, Su, arginine can significantly enhance the effect of TER on Candida albicans. Methyl sulphur, asparagus, valley, phenylpropyl, C, tryptophan can antagonize the effect of TER on Candida albicans; 6. The interaction results of amino acids and shikonin (SK) combined: cysteine, sou, ISO, semen, eggs, phenylpropyl, color, birds, Gump, silk, and alanine can significantly enhance SK The role of Candida albicans, aspartic acid and leucine, can antagonize the action of SK on Candida albicans.
The effects of 20 different amino acids on the growth of Candida albicans and the effect of interaction with different antifungal agents on the growth of Candida albicans were detected by microdilution method. The results showed that cysteine could significantly inhibit the growth of Candida albicans. The inhibitory effect of threonine on the growth of Candida albicans was weak, and other amino acids were found. The growth of Candida albicans was not inhibited. Different amino acids and antifungal drugs had different effects on the growth of Candida albicans.
In the second part, the effects of lysine, cysteine alone and combined with AmB on Candida albicans and other strains were investigated. The antifungal effect of lysine on AmB and the antifungal effect of cysteine on AmB were further investigated through the experiment of spot assay, growth curve and survival rate. The experiment selected Candida albicans as the object of study, and the use of microsatellite in the experiment. 0.5mM lysine could enhance the anti Candida albicans effect of 0.25 mu g/ml by measuring the solution based lysine; 0.25mM cysteine could inhibit the growth of Candida albicans and enhance the effect of 0.25 mu g/ml AmB on Candida albicans. Similarly, 1mM lysine could enhance 0.5 mu g/ml AmB to nalaciasa (04013803929001822090220190201309). The antifungal effect of Cryptococcus neoformans 0201309, 0.25mM cysteine could affect Candida korubica ACCT2159, Candida smooth ACCT28226, and nearly smooth Candida 22090 growth.Spot aasay experimental results showed that the lysine group had no difference with the blank group, and the consistency of 8mM lysine above and 0.25 mu g/ml AmB could inhibit the growth of Candida albicans. Long, 1mM cysteine could inhibit the growth of Candida albicans. The growth curve also showed that the combination of 1mM lysine and 0.25 mu g/ml AmB could strongly inhibit the growth of Candida albicans compared with 0.25 mu g/ml AmB. We also examined the effect of AmB and lysine on the formation of the biofilm by the XTT reduction method. The results showed that 0. The combination of 5 g/ml AmB with 4mM lysine has strong inhibition on the formation of Candida albicans biofilm and the metabolic activity of biofilm cells. At the same time, mycelium growth proves that the effect of lysine and AmB on Candida albicans biofilm may be due to the inhibition of mycelium formation.
In the third part, the synergistic mechanism of lysine and AmB was studied. The main method was the transmission electron microscope to observe the effect of two drugs on the ultrastructure of Candida albicans. Flow cytometry was used to observe the effect of the combined use of two drugs on the cell cycle, and the production of ROS in the combined cells was detected by the multi-function microplate detector. Measurement of the mitochondrial membrane potential level after the combined use of two drugs by the JC-1 kit, the effects of AmB and lysine on the oxidative damage of Candida albicans were investigated. The results showed that AmB could increase the level of endogenous reactive oxygen species in Candida albicans, while AmB and lysine were used in the combination of lysine and lysine. There was no significant difference from that in the blank group; at the same time, we also used Real-time RT-PCR to investigate the expression of redox related genes. The results showed that compared with the AmB single use group, AmB and lysine combined with ROS scavenging related gene Sod2 expression level decreased, further promoting the accumulation of endogenous ROS in Candida albicans: however, compared with the AmB single use group, The expression levels of AmB and lysine combined with oxidative stress related genes (TRR1, CaMCA1, Cap1, GRP2) were all up regulated in varying degrees, and the oxidative stress of Candida albicans increased. In addition, AmB could reduce the level of lysine in the mitochondrial membrane potential of Candida albicans, while the mitochondrial membrane potential of Candida albicans with AmB and lysine was lower than that of lysine, lysine group and lysine group. There was no obvious difference in the blank group. Compared with the AmB single use group, the membrane in the cell division of lysine and AmB group was not obvious, even disappeared, the cell cycle was blocked in the G2/M phase, and the cell mitosis was blocked. There was no significant difference between the single use group and the blank group.
In the fourth part, the results of the study in vivo showed that cysteine alone had no therapeutic effect on systemic Candida albicans in mice, but cysteine combined with amphotericin B was better than the effect of amphotericin B alone, which was consistent with the effect in vitro.
To sum up, the study found that a variety of amino acids and antifungal agents have synergistic effect in vitro; cysteine has strong antifungal activity and can enhance the antifungal activity of AmB on Candida albicans in vivo and in vitro; lysine can enhance the sensitivity of Candida albicans to AmB in vitro. The main mechanism of which is to prevent cells from the cells. The formation of the septum during the division, the G2/M phase in the cell cycle, the increase of intracellular reactive oxygen species and the decrease of the mitochondrial membrane potential, and the combination of lysine and AmB can cause some changes in the expression of redox related genes.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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