芦荟大黄素抑制金黄色葡萄球菌生物被膜形成的分子机制研究

发布时间：2018-07-27 18:54

【摘要】：金黄色葡萄球菌(Staphylococcus aureus,以下简称金葡菌)是引起人类和动物组织器官感染的主要致病菌之一。它编码大量毒力因子,可以感染生物体内不同宿主,引发多种疾病。金葡菌传播途径快、致病性强和多重耐药性等特点,使其在临床治疗上成为难点。金葡菌形成生物被膜能力强。形成的生物被膜可以降低细菌细胞代谢水平,使细菌产生屏障增强其对抗生素的敏感性,并能破坏宿主的防御系统。由于细菌耐药性问题和抗生素的不良反应逐渐增加,极大的加剧了有效化学合成药物的研发难度。因此传统中药资源为开发新型抗菌药物提供了广阔的空间。本研究通过建立S.aureus ATCC29213菌株体外生物被膜模型,鉴定其在含有3%NaCl和0.5%葡萄糖的脑心浸液肉汤(BHI)培养基中形成生物被膜能力最强。使用96孔板结晶紫染色法从几类中药单体化合物中筛选抗金葡菌生物被膜抑制剂,发现芦荟大黄素在256μg/mL时明显抑制金葡菌生物被膜形成,且微量稀释法显示其对S.aureus ATCC29213浮游菌的最小抑菌浓度(MIC)1024μg/mL。利用结晶紫染色法和荧光显微镜实验进一步考察芦荟大黄素对S.aureus ATCC29213生物被膜的作用,结果显示芦荟大黄素以剂量依赖的方式抑制金葡菌生物被膜形成,且对非生物表面生物被膜也有抑制作用。在不同时间段内加入芦荟大黄素检测其对生物被膜发育阶段的影响,发现芦荟大黄素主要作用于生物被膜初期粘附阶段和细菌增殖阶段。酶解实验结果表明,S.aureus ATCC29213在此培养条件下形成的生物被膜胞外基质的主要成分是蛋白质。通过激光共聚焦实验、自溶实验和刚果红实验进一步研究芦荟大黄素对生物被膜胞外基质的作用,结果显示芦荟大黄素可抑制胞外蛋白和胞外多糖的产生,但对胞外DNA(eDNA)几乎没有抑制作用。综上所述,本研究初步探讨了芦荟大黄素对金葡菌生物被膜抑制作用的分子机制,芦荟大黄素有望成为治疗金葡菌生物被膜感染的先导化合物,为研发新型药物奠定理论基础。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus, hereinafter referred to as Staphylococcus aureus) is one of the main pathogenic bacteria causing human and animal tissues and organs infection. It encodes a large number of virulence factors, which can infect different hosts in the organism and cause a variety of diseases. The transmission of Staphylococcus aureus is fast, the pathogenicity is strong and multiple drug resistance, so that it is in clinical practice. The biofilm formation of Staphylococcus aureus is strong. The biofilm formed to form a biofilm can reduce the metabolic level of bacterial cells, cause bacteria to produce a barrier to enhance its sensitivity to antibiotics and destroy the host's defense system. Therefore, traditional Chinese medicine resources provide a wide space for the development of new antibacterial drugs. In this study, a biological membrane model of S.aureus ATCC29213 strain in vitro was established to identify the strongest biofilm formation in the brain submerged broth (BHI) culture medium containing 3%NaCl and 0.5% glucose. It was found that the anti Staphylococcus aureus biofilm inhibitor was screened from several kinds of traditional Chinese medicine monomers. It was found that the aloe emodin inhibited the formation of the biofilm of Staphylococcus aureus at 256 g/mL, and the microdilution method showed that the minimum inhibitory concentration (MIC) of the S.aureus ATCC29213 phytoplankton was 1024 mu with crystal violet staining and fluoreme. The effect of aloe emodin to S.aureus ATCC29213 biofilm was further investigated by light microscopy. The results showed that aloe emodin could inhibit the formation of Staphylococcus aureus biofilm in a dose-dependent manner, and also inhibit the biofilm on the abiotic surface. The addition of aloe emodin to the biofilm of the biofilm in different time periods The effect of the breeding stage is that the aloe emodin mainly acts on the initial adhesion stage and the bacterial proliferation stage of the biofilm. The results of enzymatic hydrolysis show that the main component of the extracellular matrix of S.aureus ATCC29213 under this culture condition is protein. The experiment of laser coking, the autolysis experiment and the Congo red experiment are further improved. The effect of aloe emodin to extracellular matrix of biofilm was studied. The results showed that Aloe Vera could inhibit the production of extracellular and extracellular polysaccharide, but had little inhibitory effect on extracellular DNA (eDNA). In summary, the molecular mechanism of the inhibition of aloe emodin to the biofilm of Staphylococcus aureus was preliminarily discussed. Hope to become the leading compound of Staphylococcus aureus biofilm infection, and lay a theoretical foundation for the development of new drugs.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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