本文選題：產(chǎn)酶溶桿菌C3 + HSAF��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：隨著放射療法、化學(xué)療法、免疫抑制劑、抗生素和內(nèi)置醫(yī)療器械的廣泛應(yīng)用,導(dǎo)致了發(fā)生嚴(yán)重的感染,尤其是真菌感染。其中,白色念珠菌在免疫低下的病人體內(nèi)成為最易感染的真菌,也是許多重大疾病導(dǎo)致死亡的直接原因。治療白色念珠菌感染需要長(zhǎng)期大量地使用藥物,導(dǎo)致耐藥菌不斷出現(xiàn),加劇了臨床治療的難度。發(fā)現(xiàn)新結(jié)構(gòu)和新作用機(jī)制的抗真菌藥物,是控制白色念珠菌感染的重要途徑。具有活性的化合物大部分來(lái)源于微生物。長(zhǎng)期以來(lái)人們主要關(guān)注革蘭氏陽(yáng)性菌和真菌來(lái)源的活性天然產(chǎn)物,而革蘭氏陰性細(xì)菌蘊(yùn)藏的豐富天然產(chǎn)物資源沒(méi)有得到應(yīng)有的關(guān)注。近些年人們對(duì)溶桿菌的開(kāi)發(fā),得到了系列結(jié)構(gòu)新穎的活性化合物,溶桿菌正成為活性天然產(chǎn)物的新資源。本學(xué)位論文對(duì)產(chǎn)酶溶桿菌C3菌株的發(fā)酵條件進(jìn)行篩選,在其次級(jí)代謝產(chǎn)物中分離得到了4個(gè)多環(huán)四胺酸大環(huán)內(nèi)酰胺(PTM)類化合物；開(kāi)展了2個(gè)PTM類化合物的體內(nèi)外抗真菌活性評(píng)價(jià),探討了其抗真菌的作用機(jī)制。本論文第一章概述了當(dāng)前真菌感染的嚴(yán)重性和臨床抗真菌藥物品種的匱乏。實(shí)際上,目前臨床抗真菌藥物的作用靶點(diǎn)主要集中在細(xì)胞壁和細(xì)胞膜,并且導(dǎo)致大量耐藥菌株的出現(xiàn)。從新資源溶桿菌中尋找新穎的抗真菌化合物,是一條行之有效的途徑。本章綜述了溶桿菌活性天然產(chǎn)物及其生物活性和生物合成的研究現(xiàn)狀。本論文第二章是產(chǎn)酶溶桿菌C3菌株發(fā)酵培養(yǎng)條件的優(yōu)化,對(duì)該菌株中得到的代謝產(chǎn)物進(jìn)行了結(jié)構(gòu)鑒定。產(chǎn)酶溶桿菌C3菌株的平板發(fā)酵培養(yǎng)基篩選結(jié)果表明,C3菌株在TSB培養(yǎng)基中次級(jí)代謝產(chǎn)物的產(chǎn)量最高。同時(shí)我們對(duì)TSB的用量進(jìn)行考察,發(fā)現(xiàn)隨著TSB用量的減少,在C3菌株中得到的產(chǎn)物的量也相應(yīng)降低。最終我們選用1/10 TSB培養(yǎng)基進(jìn)行大規(guī)模發(fā)酵,得到4個(gè)PTM類化合物。其中,3個(gè)已知化合物(HSAF、3-deOH-HSAF和3-deOH-alteramide B)和1個(gè)新化合物alteramide B(ATB)。本論文第三章是HSAF抑制絲狀真菌和白色念珠菌的活性研究。首先以水稻稻瘟病菌為研究模型,進(jìn)行轉(zhuǎn)錄組差異分析,發(fā)現(xiàn)HSAF對(duì)細(xì)胞的凋亡途徑有重要影響。由于細(xì)胞壁具有成分比較復(fù)雜的外層和結(jié)構(gòu)比較致密的內(nèi)層結(jié)構(gòu),能夠影響小分子物質(zhì)轉(zhuǎn)運(yùn)到細(xì)胞內(nèi)。我們研究在給藥過(guò)程中小分子藥物對(duì)于細(xì)胞各組分起到的作用及其作用機(jī)制,其中首要的是獲得原生質(zhì)體,進(jìn)而以白色念珠菌為模型,在原生質(zhì)體水平,考察了HSAF作用方式。利用DCFH-DA熒光染色,檢測(cè)HSAF作用后細(xì)胞內(nèi)的活性氧(reactive oxygen species,ROS)的水平。加入四種活性氧清除劑：抗壞血酸(AA)、硫脲(TU)、乙酰半胱氨酸(NAC)和谷胱甘肽(GSH)后,HSAF的抗真菌活性明顯降低,表明HSAF通過(guò)誘導(dǎo)細(xì)胞內(nèi)ROS上調(diào)抑制真菌生長(zhǎng)。通過(guò)熒光倒置顯微鏡、流式細(xì)胞分析儀和western blotting等檢測(cè),發(fā)現(xiàn)HSAF引起線粒體膜發(fā)生變化,其電位呈現(xiàn)出下降的趨勢(shì),引起DNA損傷和細(xì)胞周期分布異常,停留在G2/M期,最終導(dǎo)致細(xì)胞發(fā)生早期和晚期凋亡。本論文第四章是新化合物ATB抗白色念珠菌的作用機(jī)制研究。ATB對(duì)白念珠菌的體外抑制較好,不僅能抑制白念珠菌酵母態(tài)的生長(zhǎng),也能抑制其菌絲體的生長(zhǎng)。ATB能夠誘導(dǎo)白色念珠菌細(xì)胞內(nèi)ROS水平的上升,引起細(xì)胞內(nèi)線粒體膜電位的下降。進(jìn)一步研究發(fā)現(xiàn),ATB能夠引起白色念珠菌細(xì)胞G2/M期停滯,導(dǎo)致細(xì)胞發(fā)生早期和晚期凋亡。AA可以抑制ATB對(duì)白色念珠菌細(xì)胞的生長(zhǎng)周期阻滯和凋亡誘導(dǎo)作用,因此,ATB通過(guò)誘導(dǎo)白色念珠菌細(xì)胞內(nèi)ROS的產(chǎn)生和積累而發(fā)揮其抗真菌活性。作用靶點(diǎn)研究結(jié)果表明,在HeLa細(xì)胞中,ATB能夠與微管蛋白結(jié)合；在體外,ATB能夠抑制微管蛋白的聚集；分子模擬表明,ATB能與白色念珠菌β-tubulin的12個(gè)關(guān)鍵氨基酸相互作用；點(diǎn)突變實(shí)驗(yàn)結(jié)果證明,ATB與β-tubulin的可能結(jié)合位點(diǎn)分別為L(zhǎng)215、L217、L273、T274和R282,其中R282是關(guān)鍵作用位點(diǎn)。小鼠體內(nèi)實(shí)驗(yàn)結(jié)果表明,ATB發(fā)揮了良好的體內(nèi)抑制白色念珠菌的作用。本論文研究發(fā)現(xiàn)了新化合物ATB,探討了其抗真菌作用靶點(diǎn)和作用機(jī)制,首次發(fā)現(xiàn)微管蛋白有望成為抗真菌藥物的新靶點(diǎn),為臨床治療白色念珠菌感染提供了新思路,對(duì)開(kāi)發(fā)新型抗真菌藥物有重要參考意義。
[Abstract]:With the extensive use of radiation therapy, chemotherapy, immunosuppressants, antibiotics, and built-in medical instruments, serious infections, especially fungal infections, are caused by Candida albicans, the most susceptible fungi in immunocompromised patients, and a direct cause of death in many major diseases. Treatment of white beads Bacteria infection requires long and large use of drugs, resulting in the continuous emergence of drug-resistant bacteria, which exacerbates the difficulty of clinical treatment. Discovering new structures and antifungal agents for new mechanisms of action is an important way to control Candida albicans infection. Most of the active compounds are derived from microorganism. The natural products of the bacteria and fungi are natural products, and the rich natural products of the Gram-negative bacteria have not been paid attention to. In recent years, a series of novel active compounds have been obtained for the development of Bacillus lysin. The bacilli have become a new resource of the active natural products. The fermentation conditions were screened and 4 polycyclic four amino acid macrocyclic amides (PTM) were isolated from its secondary metabolites, and the antifungal activity of 2 PTM compounds was evaluated in vivo and in vitro. The first chapter of this paper summarized the severity of fungal infection and the clinical antifungal infection. In fact, the targets of clinical antifungal agents are mainly concentrated in the cell walls and cell membranes and lead to the emergence of a large number of resistant strains. It is an effective way to find novel antifungal compounds from the new resources of bacilli. In this chapter, the natural products and biological activities of the bacilli are described in this chapter. The second chapter of this paper is the optimization of fermentation conditions for the strain of C3 producing Bacillus lysin. The structure of the metabolites obtained in this strain was identified. The screening results of the plate fermentation medium of C3 producing strain of Bacillus lysin showed that the secondary metabolites of the C3 strain were the highest in the TSB medium. We investigated the amount of TSB and found that as the amount of TSB decreased, the amount of products obtained in the C3 strain also decreased accordingly. Finally, we selected the 1/10 TSB medium for large-scale fermentation and obtained 4 PTM compounds. Among them, 3 known compounds (HSAF, 3-deOH-HSAF and 3-deOH-alteramide B) and 1 new compounds alteramide B. The third chapter of this thesis is the study of HSAF inhibition of filamentous fungi and Candida albicans. First of all, rice blast fungus was used as a study model to analyze the difference of transcriptional groups. It was found that HSAF had an important influence on the apoptosis pathway of cells. Molecular substances are transported to the cells. We study the role and mechanism of small molecular drugs in the process of drug delivery. The first is to obtain protoplasts, and then with Candida albicans as a model, the HSAF action formula is examined at the level of protoplast. DCFH-DA fluorescence staining is used to detect the effect of HSAF. The level of intracellular active oxygen (reactive oxygen species, ROS). After adding four kinds of active oxygen scavengers: ascorbic acid (AA), thiourea (TU), acetylcysteine (NAC) and glutathione (GSH), the antifungal activity of HSAF decreased obviously, indicating that HSAF is up to inhibit the growth of fungi by inducing the ROS up cell in the cell. By fluorescence inverted microscope, flow cells The analysis of the analyzer and Western blotting, found that HSAF caused the mitochondrial membrane changes, its potential showed a downward trend, causing DNA damage and abnormal cell cycle distribution, staying in the G2/M stage, and eventually leading to the early and late apoptosis of the cells. The fourth chapter of this paper is the study of the mechanism of the action mechanism of the new compound ATB against Candida albicans,.ATB In vitro inhibition of Candida albicans can not only inhibit the growth of Candida albicans, but also inhibit the growth of the mycelial growth of Candida albicans, which can induce the increase of ROS level in the cells of Candida albicans, causing the decrease of the mitochondrial membrane potential in the cells. Further studies have found that ATB can cause the stagnation of the G2/M phase of Candida albicans cells, leading to the refinement of the cells of Candida albicans. Early and late apoptotic.AA can inhibit the growth cycle arrest and apoptosis inducing effect of ATB on Candida albicans cells. Therefore, ATB plays its antifungal activity by inducing the production and accumulation of ROS in Candida albicans. The target point study shows that in HeLa cells, ATB can be combined with microtubule protein; In addition, ATB can inhibit the aggregation of microtubule protein; molecular simulation shows that ATB can interact with 12 key amino acids of Candida albicans beta -tubulin, and point mutation experimental results show that the possible binding sites of ATB and beta -tubulin are L215, L217, L273, T274 and R282 respectively, and R282 is the key site. TB has played a good role in inhibiting Candida albicans in vivo. In this paper, a new compound, ATB, was found to explore the target and mechanism of its antifungal action. It was found that microtubulin could be the new target of antifungal drugs for the first time. It provides a new idea for the clinical treatment of Candida albicans infection and the development of new antifungal drugs. Important reference significance.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R915

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