【作者】 高磊；

【导师】 王丽；

【作者基本信息】 吉林大学 ， 病原生物学， 2012， 博士

【摘要】 烟曲霉(Aspergillus fum gatus)是一种环境中广泛存在的腐生真菌,也是一种可经空气传播的重要条件致病性真菌。近年来,由于广谱抗生素、抗癌药物、免疫抑制剂的广泛使用,侵入性诊疗技术、器官移植的开展,艾滋病、糖尿病患者的不断增多,人口老龄化等原因,导致侵袭性肺曲霉病(Invasive pulmonary aspergillosis, IPA)的发病率急剧增加,且预后差,死亡率高达50%-95%,已成为临床上仅次于假丝酵母病的第二大真菌感染性疾病。目前,抗真菌药种类较少,毒副作用较大,价格昂贵。因此,寻找具有抗真菌能力强,毒副作用低的新型抗真菌药物,具有深远的意义。由于中药历史悠久、疗效显著、天然低毒,因而,从中药中寻找有效的抗真菌药物,促进抗真菌药物的开发,具有广阔的应用前景。一、抗烟曲霉中药单体的筛选本研究选择了20种中药的单体成分,针对7株烟曲霉临床分离株,通过微量稀释药敏法,测定药物抑菌效果,初步筛选出5种抗菌活性较好的单体,其中小檗碱的抑菌效果最佳。增加受试菌株数量后发现,小檗碱对临床分离的42株烟曲霉均有明显的抑制作用。二、小檗碱抗烟曲霉的研究1.小檗碱对烟曲霉生物学性状的影响本研究以烟曲霉IFM40808做为小檗碱抗烟曲霉作用机制的研究对象。形态学研究结果发现：肉眼观察可见小檗碱能抑制烟曲霉的菌丝生长、孢子萌发及孢子色素的生成；扫描电子显微镜观察可见小檗碱使菌丝和孢子形态发生畸变：透射电子显微镜观察可见小檗碱能破坏烟曲霉细胞壁的连续性,使细胞膜部分缺失,细胞器内容物减少。实验结果表明,小檗碱具有抑制烟曲霉的作用。2.小檗碱对烟曲霉侵袭力的影响本研究利用Transwell细胞小室,在体外模拟建立烟曲霉侵袭细胞基质模型,并探讨了小檗碱对烟曲霉孢子侵袭性的影响。通过激光共聚焦显微镜观察发现,小檗碱作用烟曲霉后,与对照组相比,在下室中FITC纳米粒子和烟曲霉菌丝体较减少,表明小檗碱抑制了烟曲霉孢子的侵袭性和生长发育。同时通过孢子侵染小鼠实验,进一步证明小檗碱处理后的烟曲霉孢子侵袭性下降,这为小檗碱预防用药提供了理论指导。本研究建立了小鼠侵袭性肺曲霉病模型,并评价了小檗碱的治疗效果。结果表明,用药后小鼠的生存率明显提高；肺脏免疫组织化学染色后发现,用药后肺内炎性反应下降；Western-blotting结果发现,药物治疗72h后,与对照组相比,小鼠的肺部炎性相关蛋白表达量下降,表明小檗碱对侵袭性肺曲霉病有较好的治疗效果。3.小檗碱与常用抗真菌药物的联合应用本研究尝试将小檗碱和常用抗烟曲霉药物联合应用。经棋盘式微量稀释实验发现,小檗碱与伊曲康唑、伏立康唑相拮抗;与5-氟胞嘧啶和特比萘芬不相关；与卡泊芬净和两性霉素B相协同。目前两性霉素B由于其毒副作用较高,限制了在临床上的应用。而通过联合用药,可以减少两性霉素B的用量,降低其毒副作用。本研究通过体内研究证实,小檗碱和两性霉素B联合应用后,可以减少两性霉素B的药物用量。这为降低两性霉素B在临床治疗中的毒副作用,提供了理论基础。4.小檗碱抗烟曲霉的作用机制本研究利用Real-time PCR、Western-blotting等分子生物学方法,发现小檗碱通过抑制烟曲霉细胞色素通路相关基因的表达,抑制了孢子色素的形成；并且可以通过抑制烟曲霉钙调磷酸酶信号通路相关基因的表达,抑制了孢子的萌发。为了探讨小檗碱抑制烟曲霉侵袭性的作用机制,本研究提取用药组和非用药组的烟曲霉分泌蛋白,分别作用于肺泡上皮细胞和支气管上皮细胞,发现细胞生长均受抑制。结果表明,小檗碱可能是通过抑制烟曲霉分泌蛋白的活性,从而导致烟曲霉孢子的侵袭性下降。由于联合药敏实验发现,小檗碱与伊曲康唑相拮抗,而后者又是抗烟曲霉的临床常用药物,同时小檗碱也较为常用,为了进一步探讨二者拮抗的机制,本研究利用Real-time PCR和高效液相色谱分析等方法,发现小檗碱可以抑制烟曲霉麦角固醇通路基因的表达,从而抑制麦角固醇的合成。这一机制与伊曲康唑的作用机制相似,这可能是两者拮抗的原因。通过以上研究表明,小檗碱是经多种途径对烟曲霉进行抑制的,这为寻找小檗碱对烟曲霉的作用靶点奠定了基础。综上所述,本研究以筛选得到的抗烟曲霉中药单体小檗碱为研究对象,通过体外、体内实验,证明了小檗碱对烟曲霉具有抑制作用,并探讨了其作用机制,这为小檗碱抗烟曲霉的临床应用提供了理论依据和技术支撑。更多还原

【Abstract】 Aspergillus fumigates is saprophyte fungi which is ubiquitous in the environment. As the airborne filamentous fungal pathogen, it is known to be a major cause of lethal lung infections in immunocompromised hosts and allergic asthma in atopic individuals. Invasive pulmonary aspergillosis has high mortality rates(50%-95%), and its incidence has been increasing gradually.Not with standing the increasing need for effective therapy, the range of antifungal agents available is limited, and some of the most effective agents are also toxic. It is necessary to find the new antifungal agents which has good anti-fungal ability and low toxicity. More and more studies showed that it is a good resources to find the antifungal agents.1. The screening of antifungal agents to Aspergillus fumigatusIn this study, we detected the efficacy of20kinds of Chinese medicine for seven Aspergillus fumigatus isolated from clinical patients by microdilution susceptibility method, determination of drug inhibitory effect, the initial screen of five kinds of better antibacterial activity of the monomer, which Berberisthe antibacterial effect of the alkali. Of berberine on42clinical isolates of Aspergillus fumigatus significantly inhibited by increasing the strain number.2. The study of Aspergillus fumigatus treated with Berberine(1) The study of Aspergillus fumigatus biological properties treated with BerberineIn this study, we selected the Aspergillus fumigatus IFM40808to study the mechanism of berberine. Morphological data showed that berberine could inhibit the growth of Aspergillus fumigatus hyphae, spore germination and spore pigment formation. Berberine can inhibit Aspergillus fumigatus hyphae and conidia form distortion by scanning electron microscope observation. In addition, berberine can destroy the continuity of the Aspergillus fumigatus cell wall, membrane partial deletion, and reduced contents of the organelle by transmission electron microscopy observation.(2) Berberine inhibited Aspergillus fumigatus invasionTo further study, we used the Transwell cell chambers established in vitro model of Aspergillus fumigatus invades the cell matrix, and invasive experiments to prove that berberine significantly inhibited the invasive A. fumigatus conidia. After the decline of Aspergillus fumigatus invasive, laser confocal microscopy, FITC nanoparticles and Aspergillus fumigatus mycelium less:in the room next to the cell chamber.We further established the invasive pulmonary aspergillosis model in mice, and treatment with berberine. Prove that berberine treatment of invasive pulmonary aspergillosis in mice survival. Using immunohistochemistry to identify the untreated group of mice lungs was further confirmed by berberine. We detected the protein expression after72h Aspergillus fumigatus infection by Western-blotting. The results showed that berberine could change in the lungs of mice of different groups on the inflammatory signaling pathways associated protein, which proved that no medication treated group inflammatory protein expression was increased in the drug treatment of inflammatory protein expression declined. At the same time, we detected the effect of mice infected with spores, and the results also support the opinion.(3) The combination of berberine with the commonly used antifungal agentsIn this study, we try to examine the effect of the antifungal agent between berberine and anti-Aspergillus fumigatus clinical drugs commonly used in combination. Our studies showed that berberine with itraconazole, voriconazole displayed antagonistic by the checkerboard microdilution experiments; berberine with5-fluorocytosine or terbinafine was no interaction; synergistic with amphotericin B and caspofungin performance. Because of the high incidence of adverse drug reactions the clinical use of amphotericin B is rather limited. With other drug since in combination a lower dose may be used, reducing the risk. This study resulted in the discovery of the theoretical combination of drugs berberine and amphotericin B. It could reduce the doses of amphotericin B. And to reduce the toxicity of amphotericin B in clinical, provides a theoretical basis.(4) The mechanisms of berberine anti-Aspergillus fumigatesWe also find that berberine can inhibit the Real-time PCR berberine can inhibit the expression of the spore pigment, which is regulated gene expression of calcineurin signaling pathways. The secreted proteins of Aspergillus fumigatus extract the drug and non-medication, the role of alveolar epithelial cells and bronchial epithelial cells, further evidence of the application of berberine treatment of Aspergillus fumigatus invasive decline. Itraconazole is the commonly used anti-Aspergillus fumigatus in clinic. Berberine is also used as anti-inflammatory in clinic. We studied the mechanism of antagonistic between berberine and itraconazole, by Real-time PCR and HPLC analysis. Our results showed that berberine could inhibit the gene expression of ergosterol pathway. This is similar to the antifungal mechanism of itraconazole. In conclusion, we selected the anti-Aspergillus fumigatus agents berberine in this study, and showed that while BER was effective in restraining the growth of Aspergillus fumigatus in vitro and in vivo. Our results proved that berberine inhibited Aspergillus fumigatus, and tried to discussed the anti-Aspergillus fumigatus mechanism of berberine. We think that our study will provied a theoretical basis and technical support for clinical application of berberine.更多还原