本文關(guān)鍵詞：龍血素A抗白念珠菌生物被膜的作用及秀麗隱桿線蟲感染模型的研究　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：白念珠菌是存在于人的粘膜表面、胃腸道和泌尿生殖道中的條件致病真菌,當(dāng)人的免疫系統(tǒng)受損時(shí)可導(dǎo)致嚴(yán)重感染的發(fā)生。許多白念珠菌感染是由于在植入的醫(yī)療裝置上,如心臟支架、尿道插管等,形成生物被膜引起的。生物被膜導(dǎo)致反復(fù)感染和對(duì)常用的抗真菌藥物的高度耐藥,因此臨床上亟需抗白念珠菌生物被膜的藥物�？拐婢腥舅幬锏难芯啃枰�?jiǎng)游锬Ｐ?但是,常用的動(dòng)物模型-小鼠感染模型成本高,實(shí)驗(yàn)周期長(zhǎng),不適用于大批量篩選藥物,此外,也不適用于批量考察白念珠菌的關(guān)鍵致病因素。因此,抗真菌感染的研究需要高通量、低成本的體內(nèi)感染模型。本課題開展了兩部分的研究,第一部分研究了龍血素A抗白念珠菌生物被膜的作用,第二部分考察了秀麗隱桿線蟲-白念珠菌感染模型在評(píng)價(jià)白念珠菌致病力和篩選抗感染藥物方面的應(yīng)用。在第一部分的研究中,我們用2,3-雙-(2-甲氧基-4-硝基-5-磺苯基)-2H-四唑-5-甲酰苯胺法篩選抗白念珠菌生物被膜的藥物時(shí),發(fā)現(xiàn)龍血素A具有較好的活性。龍血素A是從血竭中分離的苯丙素藥物,血竭是從龍血樹中獲得的樹脂。在XTT法和掃描電子顯微鏡法的實(shí)驗(yàn)中,我們發(fā)現(xiàn)≥5μg/ml龍血素A對(duì)白念珠菌生物被膜有顯著的抑制作用,而5-40μg/ml的龍血素A對(duì)白念珠菌沒(méi)有殺菌和抑菌作用。在≥5μg/ml時(shí),龍血素A能夠顯著降低細(xì)胞表面疏水性。在≥20μg/ml時(shí),龍血素A抑制液體和固體菌絲誘導(dǎo)培養(yǎng)基中的菌絲形成。同時(shí),Real Time RT-PCR結(jié)果表明龍血素A使粘附相關(guān)基因、菌絲相關(guān)基因和生物被膜相關(guān)基因的表達(dá)下調(diào)。此外,龍血素A在蠟螟感染模型和秀麗隱桿線蟲液體感染模型中顯示出體內(nèi)抗感染作用,并表現(xiàn)出較低的毒性�？偟膩�(lái)說(shuō),龍血素A表現(xiàn)出抗白念珠菌生物被膜的作用,并且這種作用可能與抑制粘附和菌絲形成相關(guān)。在第二部分的研究中,我們考察了秀麗隱桿線蟲-白念珠菌體內(nèi)感染模型,比較和評(píng)價(jià)了在液體培養(yǎng)基中和固體培養(yǎng)基上的秀麗隱桿線蟲感染模型用于考察白念珠菌致病力和藥物抗感染作用的優(yōu)勢(shì)和劣勢(shì)。首先,我們成功地重現(xiàn)了秀麗隱桿線蟲glp-4;sek-1液體感染模型,并用白念珠菌野生型SC5314、菌絲缺陷型hog1Δ/Δ、菌絲缺陷型pbs2Δ/Δ和菌絲缺陷型hst7Δ/Δ進(jìn)行秀麗隱桿線蟲glp-4;sek-1液體感染模型的實(shí)驗(yàn)證明該模型可以考察白念珠菌的致病力,發(fā)現(xiàn)白念珠菌菌絲缺陷型hog1Δ/Δ、pbs2Δ/Δ和hst7Δ/Δ感染組的生存率確實(shí)比野生型SC5314感染組的高;用抗真菌藥物-氟康唑和抗白念珠菌生物被膜藥物-龍血素A進(jìn)行秀麗隱桿線蟲glp-4;sek-1液體感染模型的實(shí)驗(yàn)證明該模型可以考察藥物的的抗真菌作用,發(fā)現(xiàn)氟康唑和龍血素A藥物治療后確實(shí)提高了秀麗隱桿線蟲的生存率;用免疫增強(qiáng)藥物-黃芪進(jìn)行秀麗隱桿線蟲glp-4;sek-1液體感染模型的實(shí)驗(yàn)檢測(cè)該模型是否可以考察藥物的免疫調(diào)節(jié)作用,但是黃芪藥物作用后沒(méi)有提高秀麗隱桿線蟲的生存率。即秀麗隱桿線蟲glp-4;sek-1液體感染模型可用于考察白念珠菌的致病力和藥物的抗真菌作用,但不能考察藥物的免疫增強(qiáng)作用。因?yàn)樾沱愲[桿線蟲glp-4;sek-1免疫缺陷,不能用于研發(fā)增強(qiáng)免疫力的藥物,所以我們嘗試用免疫功能健全的秀麗隱桿線蟲野生型N2Bristol建立液體感染模型,卻由于母體被后代幼蟲破壞而失敗。文獻(xiàn)提示,秀麗隱桿線蟲野生型N2 Bristol可用于固體感染模型考察感染過(guò)程中白念珠菌的致病力和宿主的免疫系統(tǒng)的相互作用,我們成功地重現(xiàn)了秀麗隱桿線蟲野生型N2 Bristol固體感染模型,并用白念珠菌SC5314、hog1Δ/Δ、pbs2Δ/Δ和hst7Δ/Δ進(jìn)行秀麗隱桿線蟲野生型N2 Bristol固體感染模型的實(shí)驗(yàn)檢測(cè)該模型是否可以考察白念珠菌的菌絲形成能力,但是白念珠菌hog1Δ/Δ,pbs2Δ/Δ和hst7Δ/Δ感染組與SC5314感染組的生存率沒(méi)有顯著性的差異;用氟康唑和龍血素A進(jìn)行秀麗隱桿線蟲野生型N2 Bristol固體感染模型的實(shí)驗(yàn)檢測(cè)該模型是否可以考察藥物的的抗真菌作用,但是氟康唑和龍血素A藥物治療后不能提高秀麗隱桿線蟲的生存率;用黃芪進(jìn)行秀麗隱桿線蟲野生型N2 Bristol固體感染模型的實(shí)驗(yàn)檢測(cè)該模型是否可以考察藥物的的免疫調(diào)節(jié)作用,發(fā)現(xiàn)黃芪藥物作用后確實(shí)提高了秀麗隱桿線蟲的生存率。我們推測(cè)黃芪通過(guò)提高秀麗隱桿線蟲免疫力發(fā)揮抗感染的作用。即秀麗隱桿線蟲野生型N2 Bristol固體感染模型不適用于考察白念珠菌的菌絲形成能力和藥物的抗真菌作用,可用于考察藥物的免疫調(diào)節(jié)作用�？傊�,龍血素A具有抗白念珠菌生物被膜的作用,秀麗隱桿線蟲glp-4;sek-1液體感染可用于考察白念珠菌的致病力和藥物的抗真菌作用,秀麗隱桿線蟲野生型N2Bristol固體感染模型可用于考察藥物的免疫調(diào)節(jié)作用。
[Abstract]:Candida albicans is found in the surface of mucosa, gastrointestinal and urogenital conditions of pathogenic fungi, when the person's immune system damage can lead to serious infection. Many infections are caused by Candida albicans in medical devices, such as stents, urethral catheterization, biofilm formation caused. The biofilm leads to repeated infections and antifungal drugs of highly resistant, so the clinical needs of anti Candida albicans biofilm drug. Antifungal drugs of animal models, but the small rat infected animal model - the common model of high cost, long experimental cycle, is not suitable for mass screening in addition, drugs, key pathogenic factors are also not suitable for batch inspection of Candida albicans. Therefore, the study of anti fungal infection requires high-throughput in vivo infection model of low cost. This paper carried out a study of the two part The first part, the effects of Loureirin A against Candida albicans biofilms, the second part of the study of Caenorhabditis elegans - Candida albicans infection model for screening anti infective agents in the pathogenicity of Candida albicans and evaluation. In the first part of the study, we use 2,3- bis (2- methoxy -4- nitro -5- -2H- four -5-) phenyl sulfonyl pyrazole formanilide method for screening anti Candida albicans biofilm drug discovery, Loureirin A has better activity. Loureirin A is isolated from the blood of the phenylpropanoid hormone drugs, dragon's blood is obtained from the resin in Dracaena. In XTT method and scanning electron microscopy method in the experiment, we found that more than 5 g/ml of Loureirin A on Candida albicans biofilm was significantly inhibited, and 5-40 g/ml of Loureirin A on Candida albicans has bactericidal and bacteriostatic effects. In more than 5 g/ml, Loureirin A can significantly reduce cell surface hydrophobicity . in more than 20 g/ml, Loureirin A inhibition in liquid and solid medium to induce the formation of mycelial medium. At the same time, the Real Time RT-PCR results showed that the adhesion of Loureirin A related genes, related genes and biological hyphae were down regulated expression of membrane associated genes. In addition, Loureirin A in G.mellonella infection model and Caenorhabditis elegans infection liquid showed anti infection effect in vivo models, and showed low toxicity. In general, Loureirin A exhibited anti Candida albicans biofilm effect, and this effect may be related to the inhibition of adhesion and hyphal formation. In the second part of the study, we investigated the beautiful elegans - Candida albicans infection in vivo model, comparison and evaluation of the solid medium is used to study the pathogenicity and drug resistance of Candida albicans infection effect of the advantages and disadvantages of Caenorhabditis elegans model based on infection of cultured in liquid Potential. First, we successfully reproduce the Caenorhabditis elegans glp-4; liquid SEK-1 infection model with Candida albicans wild-type SC5314, defective Hog1 Delta / delta mycelium, mycelium deficient pbs2 / delta and delta delta delta / hst7 deficient hyphae of Caenorhabditis elegans glp-4 infection model of SEK-1 liquid; experiments show that the model can investigate the pathogenicity of Candida albicans, Candida albicans hypase found defective Hog1 Delta / delta pbs2, Delta Delta and delta delta / hst7 / infection group, the survival rate is indeed better than the wild type SC5314 infection group; with antifungal drugs and fluconazole against Candida albicans biofilm drug - Loureirin A of Caenorhabditis elegans glp-4; SEK-1 liquid infection model experiments show that the model can evaluate drug antifungal effects of fluconazole and Loureirin A after drug treatment can improve the survival rate of Caenorhabditis elegans; immune enhancing drugs for Qi Huang Caenorhabditis elegans glp-4; experimental detection of SEK-1 infection model of the liquid immune model whether can the effects of drug regulation, but the effect of Radix Astragali drug did not improve survival in Caenorhabditis elegans. The Caenorhabditis elegans glp-4; SEK-1 fluid model can be used to test the pathogenicity of infection and drug resistance of Candida albicans by fungi the role, but the immune enhancement effect of drugs can not be investigated. Because of the Caenorhabditis elegans glp-4; SEK-1 immunodeficiency, enhance immunity drugs cannot be used for research and development, so we try to Caenorhabditis nematode of wild type N2Bristol with immune function to establish a complete liquid model for infection, but because the mother was offspring larvae damage and failure. The literature suggests that beautiful elegans wild-type N2 Bristol can be used for solid model to investigate the immune system infection infection and host pathogenicity of Candida albicans in the process of interaction, We successfully reproduce the Caenorhabditis elegans wild-type N2 solid Bristol infection model with Candida albicans SC5314, Hog1 / pbs2 / Delta Delta Delta, Delta and delta delta hst7 / tested Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can examine the hypha of Candida albicans bacteria forming ability however, Candida albicans Hog1 Delta / Delta, Delta / Delta and the difference of pbs2 hst7 Delta / delta infection group and SC5314 infection group, the survival rate was not significant; anti fungal effect detection of Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can evaluate drug use of fluconazole and Loureirin but A, fluconazole and Loureirin A after drug treatment can increase the survival rate of Xiu Liyin elegans; Huangqi tested Caenorhabditis elegans wild-type N2 solid Bristol infection model whether the model can evaluate drug Immunoregulation effect of Huangqi drugs found to really improve the survival rate of Caenorhabditis elegans. We speculate that Huangqi by improving the Caenorhabditis elegans immunity play a role. The ability of anti infection and drug anti fungal effect that hyphal formation in wild-type Caenorhabditis elegans N2 Bristol solid model is not suitable for investigation of infection of Candida albicans that can be used for the immunoregulation effects of drug. In short, Loureirin A has anti Candida albicans biofilm, Caenorhabditis elegans glp-4; SEK-1 infection can be used for liquid drug to investigate the pathogenicity of Candida albicans and antifungal effects of Caenorhabditis elegans wild-type N2Bristol solid model can be used to regulate infection the immune effects of drug.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285;R-332

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