本文選題：豬鏈球菌　切入點：生物被膜　出處：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：細菌生物被膜(bacterial biofilms,BF)是指單一或者多種細菌為了適應(yīng)其周邊環(huán)境吸附于生物或非生物表面,并分泌大量蛋白類、多糖類、脂類、胞外DNA等復(fù)合物,使得細菌之間相互粘連成蘑菇狀的膜樣結(jié)構(gòu)。它是與浮游態(tài)細菌相對的一種生長方式,當(dāng)細菌形成BF時,其耐藥性會增強10~1000倍,對臨床用藥挑戰(zhàn)巨大。豬鏈球菌(Streptococcus suis,S.suis)是一種重要的人畜共患病病原菌,對人和豬的健康安全危害嚴(yán)重。當(dāng)S.suis形成BF后,藥物很難完全清除,從而造成持續(xù)感染。所以,如何抑制S.suis BF的形成已成為獸醫(yī)臨床急需解決的難題。莢膜多糖(Capsular Polysaccharide,CPs)是2型S.suis的毒力因子,受莢膜多糖合成基因(cps)簇調(diào)控。它可以通過介導(dǎo)黏附影響S.suis BF的形成。cps基因簇中某些基因表達量變化或突變會導(dǎo)致莢膜多糖含量及結(jié)構(gòu)變化,從而促進細菌的黏附和BF的形成。蘆丁(Rutin)是一種黃酮類化合物,在自然界分布廣泛且價廉易得到,具有豐富的藥理作用。本課題組前期發(fā)現(xiàn)丁香葉水提物具有很好的抑制S.suis生物被膜形成的作用,通過譜效關(guān)系和灰色關(guān)聯(lián)度分析,確定蘆丁為其發(fā)揮作用的主要活性成分,但其作用機制尚不明確。本研究通過S.suis體外BF模型,應(yīng)用掃描電鏡技術(shù)和結(jié)晶紫染色法觀察蘆丁對S.suis BF的干預(yù)作用。并在S.suis BF形成的黏附階段考察蘆丁對BF形成過程中cps基因簇和毒力基因的影響,首先從基因突變層面和mRNA表達水平探討蘆丁對cps基因簇的影響,然后應(yīng)用熒光定量Real-time PCR檢測蘆丁對S.suis BF相關(guān)的毒力因子及群體感應(yīng)信號分子Lux S的影響,從黏附的角度闡明其干預(yù)豬鏈球菌生物被膜的機制。結(jié)果顯示蘆丁對S.suis的MIC為0.3125 mg/mL,蘆丁在1/2 MIC和1/4 MIC時抑制S.suis BF的形成。在1/4 MIC時,蘆丁雖然不影響S.suis的生長但能抑制其黏附。1/4 MIC蘆丁上調(diào)cps2D、cps2E、cps2Q、cps2S cps2J、cps2C、cps2R、gdh、mrp、gapdh基因mRNA的表達,下調(diào)cps2M、cps2V、cps2U、cps2B、cps2K、ef、sly基因m RNA的表達。綜上所述,蘆丁可通過調(diào)節(jié)S.suis莢膜多糖的合成以及S.suis毒力基因表達,抑制其黏附,從而抑制S.suis BF的形成,蘆丁是一種良好的黏附抑制劑。
[Abstract]:Bacterial biofilms (BFs) refers to a single or multiple bacteria that, in order to adapt to its surrounding environment, adsorb on biological or abiotic surfaces, and secrete a large number of protein, polysaccharides, lipids, extracellular DNA and other complexes.The bacteria form a mushroom-like membrane structure that adheres to each other.It is a growth pattern opposite to that of planktonic bacteria. When bacteria form BF, the drug resistance increases by 101000 times, which is a great challenge to clinical drug use.Streptococcus suis S.suis is an important zoonotic pathogen, which is harmful to human and pig health and safety.When S.suis forms BF, the drug is difficult to remove completely, causing persistent infection.Therefore, how to inhibit the formation of S.suis BF has become an urgent problem in veterinary clinic.Capsular polysaccharide (CPS) is a virulence factor of type 2 S.suis, which is regulated by CPS cluster.It can affect the formation of S.suis BF by mediating adhesion. The change or mutation of some genes in S.suis BF gene cluster will lead to changes in the content and structure of capsule polysaccharides, thus promoting the formation of bacterial adhesion and BF.Our group found that the water extract of clove leaves had a good effect on inhibiting the formation of S.suis biofilm. The main active components of Rutin were determined by spectrum and activity relationship and grey correlation analysis, but the mechanism of its action was not clear.In this study, the effect of rutin on S.suis BF was observed by scanning electron microscopy (SEM) and crystal violet staining by means of BF model of S.suis in vitro.The effects of rutin on the cps gene cluster and virulence genes during the formation of S.suis BF were investigated at the adhesion stage of S.suis BF. The effects of rutin on the cps gene cluster were investigated at the level of gene mutation and mRNA expression.Then the effects of rutin on S.suis BF related virulence factor and colony sensing signal molecule Lux S were detected by fluorescence quantitative Real-time PCR, and the mechanism of rutin interfering with the biofilm of Streptococcus suis was elucidated from the point of view of adhesion.The results showed that the MIC of rutin to S.suis was 0.3125 mg / mL. Rutin inhibited the formation of S.suis BF at 1 / 2 MIC and 1 / 4 MIC.鍦，

本文編號：1710856