本文選題：牙齦卟啉單胞菌 + 毒性因子�。� 參考：《西北大學(xué)》2013年碩士論文

【摘要】：牙齦卟啉單胞菌(Porphyromonas gingivalis,P.gingivalis]是革蘭氏陰性、嚴(yán)格厭氧菌。牙齦卟啉單胞菌產(chǎn)生多種與致病性有關(guān)的毒性因子(virulence factors),是目前已確定引起牙周炎的主要病原菌之一。近年來的研究發(fā)現(xiàn),牙齦卟啉單胞菌感染也與許多嚴(yán)重的系統(tǒng)性疾病,如動脈粥樣硬化和糖尿病等相關(guān)。研究牙齦卟啉單胞菌的致病因子作用機(jī)理及其受各種因素的調(diào)節(jié)是病原微生物學(xué)中一個重要課題。 本研究選用牙齦卟啉單胞菌低毒力菌株ATCC33277和高毒力菌株W83菌株作為研究對象,對這兩株菌部分生物學(xué)特性進(jìn)行了檢驗(yàn),研究了牙齦卟啉單胞菌的致病因子的表達(dá)受環(huán)境因素調(diào)節(jié)影響,并構(gòu)建了牙齦卟啉單胞菌的果蠅感染的致死實(shí)驗(yàn)動物模型。 牙齦卟啉單胞菌生長代謝過程受多種外界環(huán)境因素的調(diào)控,如氧化壓力、溫度、pH值、滲透壓、營養(yǎng)條件等。本實(shí)驗(yàn)中使用測定菌的生長和熒光實(shí)時定量PCR,檢測不同氧化壓力條件下、培養(yǎng)基缺失生長因子時牙齦卟啉單胞菌的生長和致病因子基因的表達(dá)變化。實(shí)驗(yàn)結(jié)果顯示H2O2在較低濃度時對生長影響不顯著,但明顯下調(diào)了毒性因子vimA、ragA、rgpA、rgpB、fimA、hagA、kgp表達(dá),且hagA、fimA基因表達(dá)量下調(diào)達(dá)10之以上。氯化血紅素或維生素K1缺乏時明顯降低ATCC33277生長,對W83生長有降低趨勢,但結(jié)果不顯著。實(shí)驗(yàn)中嘗試了使用基因同源置換的方法敲除vimA、ragA、rgpA、mfal這四個毒性基因,用于研究這些基因?qū)ιL和毒性的影響。 口腔微生物之間的相互作用可以影響生物被膜的性質(zhì)和形成。口腔菌種類繁多,除了部分致病菌外還有一些益生菌,跟致病的牙齦卟啉單胞菌等存在拮抗作用。血鏈球菌(S.scristatus)是口腔益生菌群之一,對牙齦卟啉單胞菌有抑制作用。本實(shí)驗(yàn)研究了血鏈球菌的菌體蛋白、血鏈菌素對牙齦卟啉單胞菌生長和生物被膜的影響。實(shí)驗(yàn)結(jié)論顯示血鏈球菌菌體蛋白和血鏈球菌培養(yǎng)物上清液可以提高牙齦卟啉單胞菌生物被膜產(chǎn)量,血鏈菌素對ATCC33277、W83生長抑制不明顯,但明顯降低生物被膜產(chǎn)量。 構(gòu)建動物模型是醫(yī)學(xué)和病原微生物學(xué)中研究致病性的重要方法。果蠅是常見有效的模式動物,且具有樣本量大,繁殖速度快,經(jīng)濟(jì)等優(yōu)點(diǎn),本研究中使用果蠅構(gòu)建牙齦卟啉單胞菌的動物感染模型。實(shí)驗(yàn)中使用針刺感染、喂養(yǎng)方法構(gòu)建果蠅致死模型,用于研究牙齦卟啉單胞菌致病性和毒性,以及牙齦卟啉單胞菌濃度跟致病性的聯(lián)系。結(jié)果表明牙齦卟啉單胞菌對果蠅有一定的毒性,毒性跟菌體的濃度有相關(guān)性,高度感染時可以導(dǎo)致果蠅死亡。 總之,本課題通過對ATCC33277和W83的生物學(xué)特性檢測、不同因素對致病因子的調(diào)節(jié)研究以及果蠅致死動物模型的建立,為牙齦卟啉單胞菌致病機(jī)理方面更深層次的研究,打下一定的理論和實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378

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