發(fā)布時間：2018-06-30 06:35

  本文選題：一氧化氮 + 金黃色葡萄球菌　； 參考：《中國海洋大學(xué)》2010年碩士論文

【摘要】：一氧化氮(nitric oxide, NO)是生物體內(nèi)重要的活性分子,參與了生物體內(nèi)許多重要的生理過程,例如,動物體內(nèi)的血管松弛、免疫防御、神經(jīng)傳遞以及植物體內(nèi)的種子萌發(fā)、鐵代謝、抗逆調(diào)節(jié)以及抗衰老調(diào)節(jié)等,被認(rèn)為是多功能的第二信使。NO在動物以及高等植物體內(nèi)的研究已受到廣泛的重視并取得重要進(jìn)展,然而其在細(xì)菌中,尤其是細(xì)菌形成的多細(xì)胞結(jié)構(gòu)—細(xì)菌生物被膜(biofilm)中的作用卻極少有人研究。細(xì)菌生物被膜是由細(xì)菌和其分泌的胞外基質(zhì)在物體表面形成的高度組織化的多細(xì)胞結(jié)構(gòu),是細(xì)菌產(chǎn)生抗生素耐藥和逃避機(jī)體免疫系統(tǒng)攻擊的主要原因,但其形成的分子機(jī)制比較復(fù)雜,涉及到許多的信號通路和調(diào)節(jié)蛋白,闡明細(xì)菌生物被膜形成機(jī)理對于細(xì)菌生物被膜相關(guān)感染的治療具有重要意義。實驗室前期研究發(fā)現(xiàn),NO能促進(jìn)金黃色葡萄球菌生物被膜形成,本論文主要是在實驗室前期研究的基礎(chǔ)上,對NO調(diào)節(jié)金黃色葡萄球菌生物被膜形成的作用機(jī)制進(jìn)行初步探討。 金黃色葡萄球菌致密的細(xì)胞壁結(jié)構(gòu)和大量的胞外多糖(PIA/PNAG)是影響其體內(nèi)遺傳操作的主要原因,首先利用實驗室分離得到的糖粘附素降解酶多聚β-1,6-N-乙酰葡聚糖胺裂解酶(DSPB)對金黃色葡萄球菌的電擊轉(zhuǎn)化條件進(jìn)行進(jìn)一步的優(yōu)化。PIA是β-1-6-N-乙酰胺基葡萄糖多聚物,DSPB能專一降解PIA。實驗結(jié)果表明在從限制缺陷菌株RN4220中提取質(zhì)粒過程中,加入DSPB對細(xì)胞進(jìn)行預(yù)處理可以明顯提高質(zhì)粒的純度和數(shù)量。利用0.1U的DSPB制備感受態(tài)細(xì)胞,其轉(zhuǎn)化效率相比于未處理組提高了1000倍,而且其效果優(yōu)于溶菌酶和溶葡萄球菌酶。 為了進(jìn)一步確證內(nèi)源NOS對金黃色葡萄球菌生物被膜的調(diào)節(jié)作用,利用表達(dá)載體對Saureus RN6390△nos中nos基因進(jìn)行回補,結(jié)果表明nos基因回補后回復(fù)了其生物被膜形成能力,說明內(nèi)源性NOS在金黃色葡萄球菌生物被膜形成中具有重要作用。同時,采用96孔板和Flow-cell兩種不同的生物被膜模型,通過外源加入NO供體SNP進(jìn)一步驗證了外源性NO同樣能夠促進(jìn)金黃色葡萄球菌RN6390的生物被膜形成。 根據(jù)文獻(xiàn)報道葡萄球菌生物被膜的形成方式分PIA依賴型和PIA非依賴型,PIA合成增加能促進(jìn)生物被膜的形成。那么外源NO是否是通過調(diào)節(jié)PIA的合成促進(jìn)生物被膜的形成呢?實時熒光定量PCR和胞外多糖檢測結(jié)果表明加入NO供體SNP后ica的表達(dá)量和PIA的產(chǎn)生量均降低,證明NO對S.aureus生物被膜形成的調(diào)節(jié)作用不是通過促進(jìn)PIA的產(chǎn)生而實現(xiàn)的。 S. aureusRN6390的雙組分調(diào)節(jié)系統(tǒng)srrAB與枯草桿菌ResDE雙組分系統(tǒng)高度同源,而ResDE能通過NsrR蛋白感受NO或者其衍生信號從而調(diào)節(jié)基因的表達(dá)。那么srrAB是否也能感受NO呢?定量PCR檢測顯示,生物被膜中srrAB表達(dá)量顯著提高,表明srrAB與生物被膜形成密切相關(guān)。通過同源重組構(gòu)建了S. aureusRN6390的雙組份調(diào)節(jié)系統(tǒng)srrAB的缺失突變株,檢測NO對其生物被膜形成的促進(jìn)作用是否依賴于srrAB。結(jié)果表明srrAB缺失后生物被膜形成能力降低,但NO對其生物被膜形成的促進(jìn)效應(yīng)仍然存在,因此NO對金黃色葡萄生物被膜形成的促進(jìn)與srrAB無關(guān)。 綜上,本論文在前期研究的基礎(chǔ)上初步探討了NO調(diào)節(jié)金黃色葡萄球菌生物被膜形成的作用機(jī)制,證實了這種調(diào)節(jié)作用并不依賴于能夠編碼多糖胞間粘附素的ica和雙組份系統(tǒng)srrAB。本論文揭示了NO在細(xì)菌生物被膜形成過程中的作用,并對作用機(jī)制進(jìn)行了初步探討,為進(jìn)一步的實驗提供了理論依據(jù),同時,為細(xì)菌生物被膜形成的調(diào)節(jié)機(jī)制和細(xì)菌生物被膜相關(guān)感染的預(yù)防和治療提供了新的思路。
[Abstract]:Nitric oxide (NO) is an important active molecule in the organism. It participates in many important physiological processes in the organism, such as vascular relaxation, immune defense, neural transmission, seed germination in plants, iron metabolism, anti inverse regulation and anti aging regulation in the animal body. It is considered to be the second messenger of multifunction.NO in the body. Research in animals and higher plants has received extensive attention and made significant progress. However, it is rarely studied in bacteria, especially the multicellular structure bacteria biofilm (biofilm), which is formed by bacteria. The bacterial biofilm is the height of the bacteria and its extracellular matrix on the surface of the body. The organised multicellular structure is the main reason for bacteria to produce antibiotic resistance and escape from the attack of the body's immune system. But the molecular mechanism of its formation is complex, involving many signal pathways and regulatory proteins. It is important to clarify the mechanism of bacterial biofilm formation for the treatment of bacterial biofilm related infection. The previous study found that NO could promote the formation of biofilm of Staphylococcus aureus. This paper mainly discussed the mechanism of NO regulating the formation of Staphylococcus aureus biofilm on the basis of early laboratory studies.
The dense cell wall structure of Staphylococcus aureus and a large number of extracellular polysaccharides (PIA/PNAG) are the main factors affecting the genetic manipulation in the body. First, the electrical shock conversion conditions of Staphylococcus aureus were further optimized by using the glucose adhesion degrading enzyme poly beta -1,6-N- acetylglucan lyzase (DSPB) isolated from the laboratory. .PIA is a beta -1-6-N- acetylamine based glucose polymer. DSPB can degrade PIA. experiment results show that in the process of extracting plasmids from the restricted strain RN4220, adding DSPB to the cell can obviously improve the purity and quantity of the plasmids. Using DSPB of 0.1U to prepare the receptive cells, the conversion efficiency is compared to the untreated group. It is 1000 times higher than that of lysozyme and lysostinase.
In order to further confirm the regulatory effect of endogenous NOS on the biofilm of Staphylococcus aureus, the expression vector was used to supplement the NOS gene in Saureus RN6390 delta Nos. The results showed that the NOS gene returned to its biofilm formation ability, which indicated that endogenous NOS plays an important role in the formation of Staphylococcus aureus biofilm. At the same time, using two different biofilm models of 96 orifice and Flow-cell, and adding NO donor SNP to further verify that exogenous NO can also promote the formation of biofilm of Staphylococcus aureus RN6390.
It is reported that the formation of staphylococcal biofilm is divided into PIA dependent and PIA non dependent forms. The increase of PIA synthesis can promote the formation of biofilm. Then, does exogenous NO promote the formation of biofilm by regulating the synthesis of PIA? Real time fluorescence quantitative PCR and extracellular polysaccharide detection results show ICA's table after NO donor SNP Both the amounts of PIA and the amount of NO decreased, demonstrating that the regulation of S.aureus on biofilm formation by S.aureus was not achieved by promoting the production of PIA.
The dual component system of S. aureusRN6390 is highly homologous to the two component system of Bacillus subtilis ResDE, and ResDE can regulate the expression of genes through the NO or its derived signal through the NsrR protein. Then can srrAB also feel NO? Quantitative PCR detection shows that srrAB expression in the biofilm shows that srrAB and organisms are used. The membrane formation is closely related. Through homologous recombination, the deletion mutant strain of the S. aureusRN6390 dual component regulation system srrAB is constructed, and whether the promotion of NO to its biofilm formation depends on the srrAB. result indicates that the biofilm formation ability of the srrAB is reduced after the deletion of the biofilm, but the effect of NO on the formation of its biofilm still exists, because of the existence of the effect of NO on the formation of its biofilm. The promotion of NO to the biofilm formation of Golden Grape has nothing to do with srrAB.
On the basis of previous studies, this paper preliminarily discussed the mechanism of NO regulation of the biofilm formation of Staphylococcus aureus, and confirmed that this regulation does not depend on the ICA and the dual component system srrAB. that can encode polysaccharide intercellular adhesion. This paper reveals the role of NO in the formation of bacterial biofilm. The mechanism of action is preliminarily discussed, which provides a theoretical basis for further experiments. At the same time, it provides a new way of thinking for the regulation mechanism of bacterial biofilm formation and the prevention and treatment of bacterial biofilm related infection.
【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王曉紅;桃柁酚對金黃色葡萄球菌生物膜形成的影響及其分子機(jī)制研究[D];西北農(nóng)林科技大學(xué);2012年

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本文編號：2085335