牙龈卟啉单胞菌致病性的初步研究

发布时间：2018-04-28 04:32

本文选题：牙龈卟啉单胞菌 + 毒性因子　；参考：《西北大学》2013年硕士论文

【摘要】：牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis]是革兰氏阴性、严格厌氧菌。牙龈卟啉单胞菌产生多种与致病性有关的毒性因子(virulence factors),是目前已确定引起牙周炎的主要病原菌之一。近年来的研究发现,牙龈卟啉单胞菌感染也与许多严重的系统性疾病,如动脉粥样硬化和糖尿病等相关。研究牙龈卟啉单胞菌的致病因子作用机理及其受各种因素的调节是病原微生物学中一个重要课题。本研究选用牙龈卟啉单胞菌低毒力菌株ATCC33277和高毒力菌株W83菌株作为研究对象,对这两株菌部分生物学特性进行了检验,研究了牙龈卟啉单胞菌的致病因子的表达受环境因素调节影响,并构建了牙龈卟啉单胞菌的果蝇感染的致死实验动物模型。牙龈卟啉单胞菌生长代谢过程受多种外界环境因素的调控,如氧化压力、温度、pH值、渗透压、营养条件等。本实验中使用测定菌的生长和荧光实时定量PCR,检测不同氧化压力条件下、培养基缺失生长因子时牙龈卟啉单胞菌的生长和致病因子基因的表达变化。实验结果显示H2O2在较低浓度时对生长影响不显著,但明显下调了毒性因子vimA、ragA、rgpA、rgpB、fimA、hagA、kgp表达,且hagA、fimA基因表达量下调达10之以上。氯化血红素或维生素K1缺乏时明显降低ATCC33277生长,对W83生长有降低趋势,但结果不显著。实验中尝试了使用基因同源置换的方法敲除vimA、ragA、rgpA、mfal这四个毒性基因,用于研究这些基因对生长和毒性的影响。口腔微生物之间的相互作用可以影响生物被膜的性质和形成。口腔菌种类繁多,除了部分致病菌外还有一些益生菌,跟致病的牙龈卟啉单胞菌等存在拮抗作用。血链球菌(S.scristatus)是口腔益生菌群之一,对牙龈卟啉单胞菌有抑制作用。本实验研究了血链球菌的菌体蛋白、血链菌素对牙龈卟啉单胞菌生长和生物被膜的影响。实验结论显示血链球菌菌体蛋白和血链球菌培养物上清液可以提高牙龈卟啉单胞菌生物被膜产量,血链菌素对ATCC33277、W83生长抑制不明显,但明显降低生物被膜产量。构建动物模型是医学和病原微生物学中研究致病性的重要方法。果蝇是常见有效的模式动物,且具有样本量大,繁殖速度快,经济等优点,本研究中使用果蝇构建牙龈卟啉单胞菌的动物感染模型。实验中使用针刺感染、喂养方法构建果蝇致死模型,用于研究牙龈卟啉单胞菌致病性和毒性,以及牙龈卟啉单胞菌浓度跟致病性的联系。结果表明牙龈卟啉单胞菌对果蝇有一定的毒性,毒性跟菌体的浓度有相关性,高度感染时可以导致果蝇死亡。总之,本课题通过对ATCC33277和W83的生物学特性检测、不同因素对致病因子的调节研究以及果蝇致死动物模型的建立,为牙龈卟啉单胞菌致病机理方面更深层次的研究,打下一定的理论和实验基础。
[Abstract]:Porphyromonas gingivalis P. gingivalis is Gram-negative and strictly anaerobic. Porphyromonas gingivalis produces a variety of virulence factors associated with pathogenicity and is one of the main pathogens that have been identified to cause periodontitis. Recent studies have found that Porphyromonas gingivalis infection is also associated with many serious systemic diseases, such as atherosclerosis and diabetes. It is an important subject in microbiology to study the mechanism of pathogenicity of porphyromonas gingivalis and its regulation by various factors. In this study, the low virulence strain ATCC33277 and the high virulence strain W83 of Porphyromonas gingivalis were selected as the research objects, and some biological characteristics of these two strains were tested. The expression of pathogenic factors of Porphyromonas gingivalis was studied and the lethal animal model of Drosophila gingivalis infection was established. The growth and metabolism of Porphyromonas gingivalis is regulated by many external environmental factors, such as oxidation pressure, temperature and pH value, osmotic pressure, nutritional conditions and so on. In this experiment, the growth of Porphyromonas gingivalis and the expression of pathogenicity factor gene were detected by real-time quantitative PCRs and fluorescence quantitative PCRs. The results showed that H2O2 had no significant effect on growth at lower concentration, but significantly down-regulated the expression of vimAragma rgpAbur rgpBnfgpBtfgpBFIMA, and down-regulated the expression of hagAfima gene by more than 10%. Hemin or vitamin K1 deficiency significantly decreased ATCC33277 growth and decreased W83 growth, but the results were not significant. In order to study the effects of these genes on growth and toxicity, we try to knockout four toxic genes vimAtragpArgpAfal by homologous substitution. The interaction between oral microbes can affect the properties and formation of biofilm. There are many kinds of oral bacteria, besides some pathogenic bacteria, there are some probiotics and antagonism with pathogenic porphyromonas gingivalis. Streptococcus sanguis S. scristatus is one of oral probiotic bacteria, which has inhibitory effect on Porphyromonas gingivalis. The effects of Streptococcus sanguis protein and streptomycin on the growth and biofilm of Porphyromonas gingivalis were studied. The results showed that the supernatant of Streptococcus sanguis and Streptococcus sanguis culture could increase the production of biofilm of Porphyromonas gingivalis. Streptomycin had no obvious inhibition on the growth of ATCC33277W83, but significantly decreased the production of biofilm. The establishment of animal models is an important method to study pathogenicity in medicine and pathogenic microbiology. Drosophila is a common effective model animal, and has the advantages of large sample size, rapid reproduction, economic and so on. In this study, we used Drosophila gingivalis to construct animal infection model of porphyromonas gingivalis. The lethal model of Drosophila was established by acupuncture infection and feeding to study the pathogenicity and toxicity of Porphyromonas gingivalis and the relationship between the concentration of Porphyromonas gingivalis and the pathogenicity. The results showed that Porphyromonas gingivalis had a certain toxicity to Drosophila, and the toxicity was related to the concentration of bacteria, and high infection could lead to the death of Drosophila. In conclusion, through the detection of biological characteristics of ATCC33277 and W83, the regulation of pathogenic factors by different factors and the establishment of lethal animal model of Drosophila, this study is a deeper study on the pathogenicity mechanism of Porphyromonas gingivalis. Lay a certain theoretical and experimental foundation.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378

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