烟酰胺的抗真菌增效作用及其分子机制研究

发布时间：2018-11-27 07:28

【摘要】：侵袭性真菌感染由于其高发病率和高死亡率,至今仍然是临床上的重要负担,其中白色念珠菌是导致念珠菌血症等侵袭性真菌感染疾病的主要病原体。然而现有的抗真菌药物种类少、副作用多,而且其研发速度也赶不上真菌耐药的速度,寻找新颖的抗真菌药物或现有抗真菌药物的增效剂越来越重要。烟酰胺(维生素B3)作为临床上治疗糙皮病的药物,在近年来被临床研究证实为能治疗皮肤癌、共济失调的有效药物。此外,体外实验已经证明烟酰胺能够在体外抑制多种真菌的生长,故本研究探索烟酰胺是否能作为抗真菌药物的增效剂,以期减轻临床上治疗侵袭性真菌感染的负担。药物敏感性实验显示,烟酰胺能有效增强紫草素、氟康唑、两性霉素B及卡泊芬净对白念珠菌的作用,还能减轻唑类药物的拖尾现象。棋盘式微量液基稀释法证明,烟酰胺能协同上述药物抗临床耐药的白色念珠菌、耐卡泊芬净的白色念珠菌、热带念珠菌及新生隐球菌的作用。通过构建小鼠系统性念珠菌感染的模型,联合使用烟酰胺和抗真菌药物进行考察。结果显示烟酰胺能有效增强氟康唑、卡泊芬净治疗小鼠系统性念珠菌感染的作用,使得小鼠生存率显著提高。小鼠肾脏病理切片结果证实烟酰胺与氟康唑、卡泊芬净联用还能减轻白念珠菌造成的肾脏炎症反应、菌丝侵袭等情况。烟酰胺与卡泊芬净联用的体内治疗效果呈现一定的剂量依赖性。此外,烟酰胺还能一定程度上增强两性霉素B治疗系统性念珠菌感染的作用。免疫印迹法证实,紫草素通过抑制去乙酰化相关基因HST3的作用从而诱导白念珠菌组蛋白H3上56位赖氨酸过度乙酰化,进而协同烟酰胺抗真菌,而氟康唑、两性霉素B及卡泊芬净均不能诱导H3上56位、9位及18位赖氨酸的过度乙酰化。运用流式细胞仪我们发现,烟酰胺能诱导白念珠菌一定程度的凋亡,并且能显著改变细胞周期,使得G1期或S期发生阻滞,这些作用有助于其协同抗真菌药物抗真菌。运用激光共聚焦显微镜、透射电子显微镜、絮凝试验及细胞表面疏水性实验等方法,我们发现烟酰胺通过抑制HST3的功能,从而导致白色念珠菌细胞壁上甘露糖含量减少、几丁质含量增加、β-(1,3)-glucan暴露以及细胞壁松弛、细胞膜皱缩及边缘模糊、胞浆内细胞器边缘模糊、真菌絮凝能力增强、细胞表面疏水性增加,进而协同抗真菌药物抗真菌。此外,通过微量液基稀释法筛查细胞壁相关基因缺失菌的菌库,我们发现烟酰胺对细胞壁的影响与GIN4基因密切相关。进一步的,我们通过代谢组学研究发现,烟酰胺能影响白念珠菌氨基酸代谢、三羧酸循环、氮代谢、糖酵解、磷酸戊糖途径等。特别的,烟酰胺还能导致白念珠菌胞内多胺含量的增多,而多胺与多胺合成抑制剂均能抑制烟酰胺抗白念珠菌的作用,精氨酸和鸟氨酸对烟酰胺的抗真菌作用没有显著影响,这提示多胺合成的通路中的鸟氨酸脱羧酶或其编码基因很可能在烟酰胺的增效过程中起了重要作用。综上所述,本课题研究证实烟酰胺能作为有效增强抗真菌药物治疗侵袭性真菌感染的增效剂,该增效剂通过多重机制增强抗真菌药物的作用,尤其是其抑制HST3功能从而影响细胞壁完整性的机制有助于开发新颖的抗真菌药物或抗真菌药物增效剂。
[Abstract]:Invasive fungal infection is still a clinically significant burden due to its high morbidity and high mortality, of which Candida albicans is the main pathogen of invasive fungal infection disease, such as Nostoglobinemia. However, the existing antifungal drugs have few kinds and many side effects, and the research and development speed of the antifungal medicine is not matched with the speed of the fungus resistance, and the novel antifungal medicine or the synergist of the existing antifungal medicine is more and more important. As a medicine for the clinical treatment of the rough skin disease, the bendamine (vitamin B3) has been proved to be an effective medicine for treating skin cancer and ataxia in recent years. In addition, in vitro experiments have been shown to be able to inhibit the growth of a plurality of fungi in vitro, and therefore, the present study is to explore whether or not the smoke and amine can be used as a synergist of an antifungal drug, with a view to reducing the burden on the clinical treatment of invasive fungal infections. The drug sensitivity test shows that the drug can effectively enhance the effect of the alkannin, the fluoroconrol, the amphotericin B and the capofene on the Candida albicans, and also can reduce the tailing phenomenon of the drug. The chess-type micro-liquid-based dilution method has been shown to be able to co-act with the above-mentioned drug-resistant Candida albicans, Candida albicans, Candida tropicalis, and Cryptococcus neoformans. The model of the infection of systemic candidiasis in mice was established. The results showed that the effect of flutamide on the infection of systemic candidiasis in mice was effectively enhanced, and the survival rate of mice was significantly improved. The results of the pathological section of the kidney of the mouse confirm that the combination of the phantamine with the fluconazole and the Capofene can also reduce the inflammatory reaction and the invasion of the kidney caused by the Candida albicans. The in vivo therapeutic effect of the combination of the tobacco and the Capofene showed a certain dose-dependence. In addition, the effect of amphotericin B on the treatment of systemic candidiasis is also enhanced to a certain extent. The immunoblotting method proves that the alkannin can induce the over-ethylation of the 56-position lysine on the histone H3 of the Candida albicans by inhibiting the action of the deacetylation-related gene HST3, Both amphotericin B and Capofene could not induce hyperethylation of 56, 9 and 18 lysine on H3. The flow cytometry (FCM) was used to find that a certain degree of apoptosis of Candida albicans can be induced by the amcinamide, and the cell cycle can be obviously changed, such that the G1 phase or the S phase is blocked, and these effects can help the anti-fungal drug to be anti-fungal. Using a laser confocal microscope, a transmission electron microscope, a flocculation test, a cell surface hydrophobic experiment and the like, we found that the content of mannose in the cell wall of the Candida albicans is reduced and the chitin content is increased by inhibiting the function of the HST3. (1, 3)-gluan exposure and cell wall relaxation, cell membrane shrinkage and edge blurry, cell organelle edge blurry, enhanced fungal flocculation capacity, increased cell surface hydrophobicity, and thus synergistic antifungal drug antifungal. In addition, the cell wall-related gene-deleted bacteria library was screened by a micro-liquid-based dilution method, and we found that the effect of the amantamine on the cell wall is closely related to the GIN4 gene. Further, we found that the amino acid metabolism of Candida albicans, the circulation of trivianic acid, the nitrogen metabolism, the glycolysis, the pentose phosphate pathway, and the like can be influenced by the tobacco chloroamine through the study of the metabolic group. in particular, the smoke and the amine can also lead to the increase of the content of the polyamine in the Candida albicans, This suggests that the ornithine deaminase or its coding gene in the pathway of the polyamine synthesis is likely to play an important role in the synergistic process of the amantamine. To sum up, the research of this subject confirms that the smoke and amine can be used as a synergist for effectively enhancing the treatment of the fungal infection of the invasive fungus, and the synergist is used for enhancing the action of the antifungal drug through the multiple mechanisms, in particular, that mechanism for inhibiting the function of the hst3 to affect the integrity of the cell wall is useful in the development of novel antifungal or antifungal drug potentiators.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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本文编号：2359869