本文選題：饑餓狀態(tài)　切入點(diǎn)：白色念珠菌生物膜　出處：《山東大學(xué)》2014年碩士論文

【摘要】：目的 作為人類最常見的條件致病菌——白色念珠菌(Candida albicans),所引起的感染性疾病大都是慢性和難治性的,這與其形成的生物膜結(jié)構(gòu)密切相關(guān)。白色念珠菌生物膜高耐藥性的可能機(jī)制有：生物膜基質(zhì)對藥物的空間位阻和屏障作用；生物膜中白色念珠菌的特殊生長狀態(tài),例如遲緩生長狀態(tài)；基因水平的耐藥機(jī)制；生物被膜內(nèi)細(xì)胞質(zhì)膜脂質(zhì)成分的影響；以及近幾年提出的‘滯留菌’的產(chǎn)生等等。越來越多的研究提示,正是滯留菌(Persisters)的存在,使得藥物無法完全清除白色念珠菌生物膜,對慢性遷延性感染的產(chǎn)生起到了關(guān)鍵性的作 用。 已有研究證實(shí)了細(xì)菌滯留菌的產(chǎn)生機(jī)制主要包括以下2個(gè)方面：微生物群體中生長速度的異質(zhì)性,極少數(shù)生長處于靜止?fàn)顟B(tài)的菌細(xì)胞亞群成為滯留菌；饑餓等環(huán)境因素導(dǎo)致的SOS反應(yīng),即極端不利環(huán)境因素誘導(dǎo)。這表明滯留菌的產(chǎn)生機(jī)制,并非單一的由生長狀態(tài)異質(zhì)性造成的,而是與環(huán)境因素的誘導(dǎo)也有密切關(guān)系。那么,改變生物膜代謝的營養(yǎng)環(huán)境,是不是也會對白色念珠菌耐藥滯留菌的產(chǎn)生有一定的影響呢? 本實(shí)驗(yàn)即通過研究饑餓環(huán)境對白色念珠菌生物膜以及其耐藥滯留菌形成的影響,證實(shí)白色念珠菌生物膜耐藥滯留菌形成的第二種機(jī)制(環(huán)境因素影響機(jī)制)；并且,進(jìn)一步運(yùn)用基于Taqman探針的單細(xì)胞real-time RT-PCR技術(shù)相對定量檢測滯留菌相關(guān)基因,為今后白色念珠菌耐藥滯留菌相關(guān)基因的研究提供方便、可行的研究技術(shù)。 材料和方法 (1)饑餓狀態(tài)對白色念珠菌生物膜及其耐藥滯留菌形成的影響 體外分別構(gòu)建對數(shù)期、穩(wěn)定期和饑餓狀態(tài)白色念珠菌生物膜模型,兩性霉素B(AmB)沖擊作用24h,沖擊前后均進(jìn)行活菌菌落計(jì)數(shù)(CFU),計(jì)算得出滯留菌比例并進(jìn)行統(tǒng)計(jì)學(xué)分析,同時(shí)通過共聚焦顯微鏡觀察和分析各組生物膜的形成狀況。 (2)基于Taqman探針的單細(xì)胞real-time RT-PCR技術(shù)相對定量檢測滯留菌相關(guān)基因的表達(dá)水平 倒置顯微鏡下,使用自行拉制的分離微吸管吸取非滯留菌細(xì)胞和滯留菌細(xì)胞,進(jìn)行基于Taqman探針的單細(xì)胞real-time RT-PCR,相對定量檢測白色念珠菌生物膜耐藥滯留菌EFG1、ERG2、MKC1、GIN4基因的表達(dá)水平,采用SPSS11.70軟件包對數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 (1)對數(shù)期、穩(wěn)定期和饑餓狀態(tài)白色念珠菌生物膜的形成能力依次下降(P0.05),具有顯著性差異,對數(shù)期和穩(wěn)定期白色念珠菌生物膜形成的為菌絲相生物膜,而饑餓態(tài)白色念珠菌形成的為酵母相生物膜。藥物作用后滯留菌比例依次上升(P0.05)。 (2)基于Taqman探針的單細(xì)胞real-time RT-PCR技術(shù)相對定量地檢測出了白色念珠菌滯留菌中EFG1、ERG2、MKC1、GIN4的表達(dá)水平。其中,除了MKC1在滯留菌細(xì)胞和非滯留菌細(xì)胞中的表達(dá)水平無顯著性差異(P0.05)外,EFG1、 ERG2、GIN4在滯留菌中的表達(dá)水平明顯升高(P0.05),具有顯著性差異。 結(jié)論 (1)營養(yǎng)狀態(tài)對白色念珠菌滯留菌的產(chǎn)生有影響,饑餓狀態(tài)能夠提高滯留菌的產(chǎn)生比例,提示白色念珠菌耐藥滯留菌形成與營養(yǎng)狀態(tài)等環(huán)境因素有關(guān)。 (2)自制的分離微吸管能夠成功分離出滯留菌細(xì)胞,在此基礎(chǔ)上,基于Taqman探針的單細(xì)胞real-time RT-PCR技術(shù)是快速檢測滯留菌相關(guān)基因表達(dá)水平的有效方法,為今后研究白色念珠菌滯留菌相關(guān)基因提供了方便、可行的研究技術(shù)。 (3)白色念珠菌滯留菌的形成與基因EFG1、ERG2以及GIN4均密切相關(guān),并非由單一的信號通路決定的,受多條信號途徑的調(diào)節(jié)。
[Abstract]:objective
As one of the most common human opportunistic pathogen, Candida albicans (Candida albicans), infectious diseases caused by mostly chronic and refractory, closely related with the formation of the biofilm structure. The possible mechanism of Candida albicans biofilm high resistance: space biofilm matrix of drug resistance and barrier special effects; growth state in biofilms of Candida albicans, such as slow growth state; mechanism of resistance gene level; biological effect of cytoplasmic membrane lipid composition in the film; and put forward in recent years' persisters' production and so on. More and more studies suggest that it is the presence of persisters (Persisters) the drug, unable to completely remove the Candida albicans biofilm in chronic persistent infection have played a key role
Use.
Studies have confirmed the mechanism of bacterial retention of bacteria mainly include the following 2 aspects: the heterogeneity of the growth rate of the microbial population growth, a handful of quiescent cell subsets become stranded bacteria; SOS reaction led to starvation and other environmental factors, in which the extreme adverse environmental factors. This indicates that the mechanism of induced retention of bacteria that is not a single state of growth caused by heterogeneity, induced and environmental factors but also have close relationship. So, nutrition environment change membrane metabolism, is not also on Candida albicans resistance retention producing bacteria have certain effect?
Effect of this experiment through the study of starvation on Candida albicans biofilm and the resistance of persisters formation, second mechanisms that Candida albicans biofilm formation (resistant bacteria retention effect mechanism of environmental factors); and further use of the relative quantitative detection of single cell real-time RT-PCR Taqman probe persisters related genes based on convenience for the future study of Candida albicans resistant bacteria retention related genes, the research technique is feasible.
Materials and methods
(1) the effect of starvation on the formation of Candida albicans biofilm and its drug-resistant stranded bacteria
In vitro constructed logarithmic phase, stable phase and starvation of Candida albicans biofilm model, amphotericin B (AmB) 24h impact, the impact of both before and after the live bacteria colony count (CFU), calculated the proportion of persisters were statistically analyzed and observed by confocal microscopy and analysis of each biofilm formation.
(2) single cell real-time RT-PCR technique based on Taqman probe to quantitatively detect the expression level of retained bacteria related genes
Under the microscope, using separate self drawn micro Straw from non persister cells and retention of bacterial cells, Taqman probe for single cell real-time RT-PCR based on the relative quantitative detection of Candida albicans biofilm drug resistance bacteria EFG1 ERG2, MKC1 retention, and the expression level of GIN4 gene, package the data were analyzed using SPSS11.70 software.
Result
(1) the logarithmic phase, stable phase and starvation formation of Candida albicans biofilm decreased (P0.05), with a significant difference, log phase and stable phase of Candida albicans biofilm formation for hyphal biofilm, and the hunger for the formation of Candida albicans biofilm. The yeast phase after the drug retention of bacteria in order to rise the proportion (P0.05).
(2) single cell real-time RT-PCR technology of Taqman probe quantitative to detect Candida albicans bacteria retention based on ERG2, EFG1, MKC1, the expression level of GIN4. Among them, in addition to MKC1 was significantly higher in the persister cells and non expression level differences in bacterial cell retention (P0.05), EFG1, ERG2. The expression level of GIN4 in residence in bacteria increased significantly (P0.05), with a significant difference.
conclusion
(1) nutritional status has an effect on the production of Candida albicans, and starvation can increase the proportion of the remaining bacteria, indicating that the occurrence of drug-resistant bacteria in Candida albicans is related to environmental factors such as nutritional status.
(2) the self-made micro Straw separation can be successfully isolated stranded bacterial cells, on this basis, based on the single cell real-time RT-PCR technology of Taqman probe is rapid detection of persisters related gene expression level of effective methods, for the future study of Candida albicans persisters related gene research provides a convenient, feasible technology.
(3) the formation of Candida albicans is closely related to the genes EFG1, ERG2 and GIN4. It is not determined by a single signaling pathway, but regulated by multiple signaling pathways.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.54

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本文編號：1674244