【作者】 李妍；

【导师】 娄红祥；

【作者基本信息】 山东大学 ， 天然药物化学， 2012， 博士

【摘要】 近年来,由于多种高效、广谱抗菌药物的大量使用,器官移植工作的广泛开展,激素及免疫抑制剂的应用,血液恶性肿瘤患者中抗肿瘤药物的应用,各种人工植入性方法的操作,如人造关节、心脏瓣膜置换、中心静脉插管等,真菌感染的发病率呈逐年增多的趋势。同时,药物的选择性压力迫使微生物产生了多种耐药机制以保证物种的延续,其中,微生物产生生物被膜(biofilm)就是一种重要的耐药机制。植入性器材,比如各种人工假体、血管内插管等常常发生真菌生物被膜感染。微生物形成生物被膜之后,耐药性可发生极为显著的变化,如白色念珠菌(Candida albicans)形成生物被膜之后,对氟康唑的耐药性成倍增加。如何防治和克服C. albicans生物被膜耐药问题,已成为临床上极具挑战性的课题。天然药物仍然是治疗多种疾病的重要来源。苔藓类植物作为传统药用植物,被应用于多种疾病的治疗,如心血管疾病、支气管炎、皮肤病等,近年来,植物学家从苔藓植物中分离了大量结构新颖且活性显著的萜类和芳香族化合物,其中许多可作为新药研究的优良先导化合物。苔藓类中的地钱属植物为叶状体苔类,从中分离获得的大环双联苄类化合物具有广泛的生物学活性,如抗氧化、抗真菌、抗病毒、抗细菌和细胞毒活性等,具有极高的研究价值。Riccardin D (RCD)是从地钱(Marchantia polymorpha L.(Marchantiaceae))中分离得到的一种大环双联苄类化合物,结构上以C6-C2-C6为母核,通过C-O-C醚桥和(／或)C-C键连接而成。进一步的研究发现,RCD不仅具有抗C. albicans的作用,而且对于C. albicans于体外形成的生物被膜有一定抑制作用,分子机制研究提示,RCD的抗C. albicans生物被膜活性是通过阻碍菌丝形成而实现的。RCD对于C. albicans生物被膜的清除能力、药敏特性,及与其他抗真菌药物联用时是否具协同作用,对于体内形成的生物被膜作用如何,尚需进一步的系统研究。本课题应用C. albicans生物被膜感染的体外和体内模型,观察了被膜形成不同阶段的形态学特征。应用此模型,研究了念珠菌在形成生物被膜前后对于实验药物RCD单用或与其他抗真菌药物联合应用时的药敏性质特征,比较不同方法用于药敏试验结果评价的适宜性；采用“抗生素封管”给药方法,利用生物被膜体内外模型进行RCD药效学研究,包括药物单用及与氟康唑联合应用时的抗念珠菌效果,应用平板计数法评价试验药物对于体内念珠菌被膜的抑制作用,并采用扫描电镜、激光共聚焦扫描显微镜等方法观察药物作用前后生物被膜的形态学改变；选择菌体黏附与菌丝生长相关基因,制备相应的引物和探针,采用RT-PCR法,考察RCD作用前后有关基因表达的变化,推测RCD抑制念珠菌被膜生长的分子学机制。课题首先构建了体外载体材料附着生物被膜模型和体内中心静脉插管相关性C. albicans生物被膜感染动物模型,研究了被膜的生长动力学、被膜生长的影响因素与微观形态学特征。在被膜生长前期,菌细胞的含量随时间的延长而明显增多,被膜形成24h时,菌落计数达106CFU,之后生长较前趋于缓慢。被膜生长与菌种和所黏附材料表面特性有关。其微观形态因生长阶段的不同而有明显不同,主要表现在菌体黏附量的多少、菌丝与假菌丝形成及胞外基质等方面的差异。课题同时研究了C. albicans不同菌株ATCC10231、SC5314、YEM30和CA10在形成被膜前后对于试验药物RCD的敏感性,被膜形成之后,其耐药性增加,增加程度与菌种有关。当RCD与抗真菌药物氟康唑、两性霉素B、卡泊芬净分别联用时,对于各试验菌株形成生物被膜前后有不同程度的增敏作用。药物联用效果以两种方法评价,分别是基于LA理论的FICI法和基于BI理论的△E法,两种方法评价药物抗游离菌作用的结果相符率为75%。采用抗菌药物封锁试验方法,将不同浓度(8,16,32,64μg/ml)的RCD药液于体外连续作用1、3、5、7日,考察药物对念珠菌成熟生物被膜的清除效果。菌落计数结果证实,RCD对于生长24h的C. albicans生物被膜有抗菌效果,且药物作用时间越长,浓度越高,其抑制被膜生长的强度就越强。以评价被膜中细胞活力为目的的形态学试验亦证明,药物浓度的增加与作用时间的延长可降低被膜中细胞的活力,甚至将被膜完全清除。试验同时考察了RCD与FLC联合应用时对于体外C. albicans生物被膜的抑制作用。结果显示,与FLC单用相比,RCD与FLC联用组生物被膜的菌落计数显著降低,提示RCD对FLC的抗念珠菌生物被膜有增效作用。课题利用插管相关性念珠菌生物被膜感染动物模型,考察了试验药物对于体内形成的念珠菌生物被膜的药效。模型建立后,在生物被膜形成的不同时段给药,分别考察RCD对于生物被膜的预防和清除作用,应用菌落计数法和形态学观察法进行评价。结果显示,在被膜形成早期(插管感染4h)给予RCD,并在插管内保留8小时,感染24小时后取标本观察,药物可有效预防被膜的形成,与对照组相比,给药组菌量显著降低,其抑菌效应呈剂量依赖性。将RCD与氟康唑联用,可增加后者对于体内被膜形成的预防效果。在插管感染24h时给予不同浓度的RCD,使之在管内每日保留8h,持续作用5日,药物对于生长24h的生物被膜同样有呈剂量依赖性的抑制作用,扫描电镜的微观形态观察证实了菌落计数结果。为进一步研究RCD于体内外抑制C. albicans生物被膜的内在机制,设计RT-PCR试验,分别取体内和体外试验中RCD作用前后的生物被膜标本,选择与菌体黏附和菌丝生长相关的基因ALS1, ALS3, ECE1, EFG1, HWP1及上游调节基因CDC35进行扩增,并与不加药组标本作对照。对于体内外标本的PCR检测结果表明,RCD作用后,上述基因的表达均呈下调趋势,在体外试验中,以HWP1下调最为显著,体内外结果有较好的一致性。这一结果提示RCD通过抑制菌体黏附和菌丝形成而对C. albicans被膜生长产生抑制作用。更多还原

【Abstract】 In recent years, the extensive use of efficient and wide-spectrum antimicrobial agents, immunosuppressive agents in organ transplant recipients, antineoplastics in patients with hematologic malignancies, the operation of artificial implantation, such as nearthrosis, heart valve replacement and central venous catheter, contributed to the increasing morbidity associated with fungal infections. The selective pressure of antimicrobials forced microorganisms produced a variety of mechanisms of drug resistance to ensure the continuation of the species, among which biofilms formation is one of the most important drug resistance mechanisms. Fungal biofilms associated infections often occurs when implanting materials are used, such as artificial prosthesis, venous catheters, et al. The susceptibility of microorganisms against antimicrobial agents may change a lot after biofilms formation. For example, Candida albicans biofilms are significantly less susceptible than in planktonic state to antifungals. How to prevent and manage the drug resistance of C. albicans biofilms has become a clinically challenging task.Natural products are still major sources of innovative therapeutic agents in various conditions. Liverworts have been used in some remedies of folk medicine to treat illnesses of the cardiovascular system, bronchitis and skin diseases as well. In the past decades, various types of lipophilic terpenoids and aromatic compounds showing significant biological activities have been isolated from the bryophytes, some of which can be used as good lead compounds for new drug development. Macrocyclic bis(bibenzyls) compounds isolated from liverworts Marchantia were found to have a wide range of biological activities, such as antioxidation, antifungi, antivirus, antibacteria and cytotoxicity, and have a high value for study. Riccardin D (RCD), a macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L.(Marchantiaceae), structurally consisted of two biphenyl bonds linked by diaryl ether bonds and phenolic hydroxyl groups in different numbers. Further investigation found that RCD had the antifungal effect not only against C. albicans, but against C albicans biofilms formed in vitro. Molecular mechanism study revealed that RCD inhibit C. albicans biofilms via retarding hyphae formation. The biofilms clearance effect, susceptibility property, the antifungal efficacy used with other antifungal agents, and the anti-biofilms formed in vivo need to be further studied.The study established in vitro and in vivo C. albicans biofilms-associated infective model, and observed the morphology at different developing stages of biofilms growth. Using this model, Candida biofilms formed in vitro and in vivo were studied, including susceptibility property of C. albicans in biofilms or as planktonic cells against RCD when used alone or in combination with other antifungal agents. Different methods were applied to estimate the antifungal effect. RCD solutions at different concentrations were administered locally by using "antibiotics lock technique" to the infective catheters, using quantitative method of colony culture to assess the inhibition efficacy of experimental agents against C. albicans biofilms in vivo. Scanning electron microscopy and confocol laser scanning microscopy were used to evaluate the morphology of C. albicans biofilms before and after drug challenge. Colony culture and morphology observation were used to assess the effect of RCD used alone or in combination with fluconazole on C. albicans biofilms. Quantitative real-time reverse transcription-PCR (RT-PCR) was used to compare mRNA abundances of the genes related to fungal adherence and hypha growth. The TaqMan MGB probe and primer sets were designed for the target genes and the control gene. The molecular mechanism of the inhibition effect of RCD on C. albicans biofilms was speculated according to the change of genes expression before and after RCD challenge.The study developed in vitro bio-material adherence C. albicans biofilms infective model and CVC-related animal model, and investigated the growth kinetics, impact factors on biofilms development and the micro-morphology properties. During the early phase of biofilms growth, fungal cells increased dramatically. Colony counting was about106CFU when biofilms grew for24h, and afterwards the cell counts increased slowly. Biofilms formation related to the nature of adherence materials and microorganism isolations. Morphology of biofilms changed significantly as time went on, mainly embodied in cell counts, hypha formation and amount of extracellular matrix.The susceptibility of C. albicans isolates ATCC10231、SC5314、YEM30and CA10in both planktonic and biofilms state against experimental agents was also investigated. Results showed that the resistance of C. albicans increased exponentially after biofilms formation. When RCD were used in combination with fluconazole, amphotericin B or caspofungin, it showed synergistic or indifferent effect against various isolates either in planktonic cells or in biofilms. We applied FICI method based on LA theory and△E method based on BI theory to evaluate the antifungal effect when RCD was used with other agents. The accordance ratio of the two methods was75%when used in assessing the antifungal effect on planktonic cells.RCD solutions at different concentrations (8,16,32,64μg/ml) were administered to C. albicans biofilms formed in vitro using antibiotic lock technique for1,3,5or7days continuously to investigate the efficacy of anti-biofilms of RCD. Colony counting proved that RCD showed antifungal effect against24-h-old biofilms. The strength of antifungal effect was related to the drug duration and concentrations. Morphology experiment which aimed to assess cell viability also proved that the increase of drug concentration and duration could decrease the cell viability of the biofilms, even some of them was eliminated totally. The anti-biofilms effect of RCD when combined with fluconazole was studied. Results showed that colony counting in combination group decreased significantly than the that challenged by FLC alone, suggesting RCD had hypersensitivity effect to fluconazole against C. albicans biofilms.Using catheter related C. albicans biofilms infective animal model, the anti-biofilms of experimental drugs. After rabbit model development, RCD solutions were administered at different biofilms formation phase, and evaluated the prevention and therapeutic effect of RCD against C. albicans biofilms. The results were assessed by colony counting and morphology observing. The specimen were taken out after24hours from the initiation of infection. Data showed that RCD could prevent the biofilms formation dose dependently when administered after4h of biofilms growth and maintained in the catheters for8hours. Compared with the control group, colony counting in RCD-challenged group decreased significantly. RCD could increase the prevention effect of fluconazole on C. albicans biofilms according to the results of drug combination experiment. When administered to the24-h-old biofilms at different concentrations and maintained for8hours per day for5days continuously, RCD showed inhibition effect in a dose dependent manner. Micro-morphology observation of scanning electron microscope proved the colony counting results.We designed RT-PCR experiment to investigate the molecular mechanism of the inhibition effect of RCD against C. albicans biofilms. Specimens of C. albicans biofilms formed in vitro and in vivo were collected and genes related to cells adherence and hypha growth ALS1, ALS3, ECE1, EFG1, HWP1and the upstream regulating gene CDC35were selected and amplified. Biofilms not treated by RCD worked as comparison. Results showed that the genes expression were all down-regulated. The expression of HWP1was decreased most significantly in in vitro study, and there was good accordance between in vitro and in vivo experiment. This suggested that RCD inhibited the biofilms growth via fungal cells adherence and hypha formation.更多还原