蘆薈大黃素抑制金黃色葡萄球菌生物被膜形成的分子機(jī)制研究
發(fā)布時(shí)間：2018-07-27 18:54
【摘要】：金黃色葡萄球菌(Staphylococcus aureus,以下簡(jiǎn)稱金葡菌)是引起人類和動(dòng)物組織器官感染的主要致病菌之一。它編碼大量毒力因子,可以感染生物體內(nèi)不同宿主,引發(fā)多種疾病。金葡菌傳播途徑快、致病性強(qiáng)和多重耐藥性等特點(diǎn),使其在臨床治療上成為難點(diǎn)。金葡菌形成生物被膜能力強(qiáng)。形成的生物被膜可以降低細(xì)菌細(xì)胞代謝水平,使細(xì)菌產(chǎn)生屏障增強(qiáng)其對(duì)抗生素的敏感性,并能破壞宿主的防御系統(tǒng)。由于細(xì)菌耐藥性問題和抗生素的不良反應(yīng)逐漸增加,極大的加劇了有效化學(xué)合成藥物的研發(fā)難度。因此傳統(tǒng)中藥資源為開發(fā)新型抗菌藥物提供了廣闊的空間。本研究通過建立S.aureus ATCC29213菌株體外生物被膜模型,鑒定其在含有3%NaCl和0.5%葡萄糖的腦心浸液肉湯(BHI)培養(yǎng)基中形成生物被膜能力最強(qiáng)。使用96孔板結(jié)晶紫染色法從幾類中藥單體化合物中篩選抗金葡菌生物被膜抑制劑,發(fā)現(xiàn)蘆薈大黃素在256μg/mL時(shí)明顯抑制金葡菌生物被膜形成,且微量稀釋法顯示其對(duì)S.aureus ATCC29213浮游菌的最小抑菌濃度(MIC)1024μg/mL。利用結(jié)晶紫染色法和熒光顯微鏡實(shí)驗(yàn)進(jìn)一步考察蘆薈大黃素對(duì)S.aureus ATCC29213生物被膜的作用,結(jié)果顯示蘆薈大黃素以劑量依賴的方式抑制金葡菌生物被膜形成,且對(duì)非生物表面生物被膜也有抑制作用。在不同時(shí)間段內(nèi)加入蘆薈大黃素檢測(cè)其對(duì)生物被膜發(fā)育階段的影響,發(fā)現(xiàn)蘆薈大黃素主要作用于生物被膜初期粘附階段和細(xì)菌增殖階段。酶解實(shí)驗(yàn)結(jié)果表明,S.aureus ATCC29213在此培養(yǎng)條件下形成的生物被膜胞外基質(zhì)的主要成分是蛋白質(zhì)。通過激光共聚焦實(shí)驗(yàn)、自溶實(shí)驗(yàn)和剛果紅實(shí)驗(yàn)進(jìn)一步研究蘆薈大黃素對(duì)生物被膜胞外基質(zhì)的作用,結(jié)果顯示蘆薈大黃素可抑制胞外蛋白和胞外多糖的產(chǎn)生,但對(duì)胞外DNA(eDNA)幾乎沒有抑制作用。綜上所述,本研究初步探討了蘆薈大黃素對(duì)金葡菌生物被膜抑制作用的分子機(jī)制,蘆薈大黃素有望成為治療金葡菌生物被膜感染的先導(dǎo)化合物,為研發(fā)新型藥物奠定理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61
本文編號(hào)：2148827
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61
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