煙酰胺的抗真菌增效作用及其分子機(jī)制研究
發(fā)布時(shí)間：2018-11-27 07:28
【摘要】：侵襲性真菌感染由于其高發(fā)病率和高死亡率,至今仍然是臨床上的重要負(fù)擔(dān),其中白色念珠菌是導(dǎo)致念珠菌血癥等侵襲性真菌感染疾病的主要病原體。然而現(xiàn)有的抗真菌藥物種類(lèi)少、副作用多,而且其研發(fā)速度也趕不上真菌耐藥的速度,尋找新穎的抗真菌藥物或現(xiàn)有抗真菌藥物的增效劑越來(lái)越重要。煙酰胺(維生素B3)作為臨床上治療糙皮病的藥物,在近年來(lái)被臨床研究證實(shí)為能治療皮膚癌、共濟(jì)失調(diào)的有效藥物。此外,體外實(shí)驗(yàn)已經(jīng)證明煙酰胺能夠在體外抑制多種真菌的生長(zhǎng),故本研究探索煙酰胺是否能作為抗真菌藥物的增效劑,以期減輕臨床上治療侵襲性真菌感染的負(fù)擔(dān)。藥物敏感性實(shí)驗(yàn)顯示,煙酰胺能有效增強(qiáng)紫草素、氟康唑、兩性霉素B及卡泊芬凈對(duì)白念珠菌的作用,還能減輕唑類(lèi)藥物的拖尾現(xiàn)象。棋盤(pán)式微量液基稀釋法證明,煙酰胺能協(xié)同上述藥物抗臨床耐藥的白色念珠菌、耐卡泊芬凈的白色念珠菌、熱帶念珠菌及新生隱球菌的作用。通過(guò)構(gòu)建小鼠系統(tǒng)性念珠菌感染的模型,聯(lián)合使用煙酰胺和抗真菌藥物進(jìn)行考察。結(jié)果顯示煙酰胺能有效增強(qiáng)氟康唑、卡泊芬凈治療小鼠系統(tǒng)性念珠菌感染的作用,使得小鼠生存率顯著提高。小鼠腎臟病理切片結(jié)果證實(shí)煙酰胺與氟康唑、卡泊芬凈聯(lián)用還能減輕白念珠菌造成的腎臟炎癥反應(yīng)、菌絲侵襲等情況。煙酰胺與卡泊芬凈聯(lián)用的體內(nèi)治療效果呈現(xiàn)一定的劑量依賴(lài)性。此外,煙酰胺還能一定程度上增強(qiáng)兩性霉素B治療系統(tǒng)性念珠菌感染的作用。免疫印跡法證實(shí),紫草素通過(guò)抑制去乙酰化相關(guān)基因HST3的作用從而誘導(dǎo)白念珠菌組蛋白H3上56位賴(lài)氨酸過(guò)度乙�；�,進(jìn)而協(xié)同煙酰胺抗真菌,而氟康唑、兩性霉素B及卡泊芬凈均不能誘導(dǎo)H3上56位、9位及18位賴(lài)氨酸的過(guò)度乙酰化。運(yùn)用流式細(xì)胞儀我們發(fā)現(xiàn),煙酰胺能誘導(dǎo)白念珠菌一定程度的凋亡,并且能顯著改變細(xì)胞周期,使得G1期或S期發(fā)生阻滯,這些作用有助于其協(xié)同抗真菌藥物抗真菌。運(yùn)用激光共聚焦顯微鏡、透射電子顯微鏡、絮凝試驗(yàn)及細(xì)胞表面疏水性實(shí)驗(yàn)等方法,我們發(fā)現(xiàn)煙酰胺通過(guò)抑制HST3的功能,從而導(dǎo)致白色念珠菌細(xì)胞壁上甘露糖含量減少、幾丁質(zhì)含量增加、β-(1,3)-glucan暴露以及細(xì)胞壁松弛、細(xì)胞膜皺縮及邊緣模糊、胞漿內(nèi)細(xì)胞器邊緣模糊、真菌絮凝能力增強(qiáng)、細(xì)胞表面疏水性增加,進(jìn)而協(xié)同抗真菌藥物抗真菌。此外,通過(guò)微量液基稀釋法篩查細(xì)胞壁相關(guān)基因缺失菌的菌庫(kù),我們發(fā)現(xiàn)煙酰胺對(duì)細(xì)胞壁的影響與GIN4基因密切相關(guān)。進(jìn)一步的,我們通過(guò)代謝組學(xué)研究發(fā)現(xiàn),煙酰胺能影響白念珠菌氨基酸代謝、三羧酸循環(huán)、氮代謝、糖酵解、磷酸戊糖途徑等。特別的,煙酰胺還能導(dǎo)致白念珠菌胞內(nèi)多胺含量的增多,而多胺與多胺合成抑制劑均能抑制煙酰胺抗白念珠菌的作用,精氨酸和鳥(niǎo)氨酸對(duì)煙酰胺的抗真菌作用沒(méi)有顯著影響,這提示多胺合成的通路中的鳥(niǎo)氨酸脫羧酶或其編碼基因很可能在煙酰胺的增效過(guò)程中起了重要作用。綜上所述,本課題研究證實(shí)煙酰胺能作為有效增強(qiáng)抗真菌藥物治療侵襲性真菌感染的增效劑,該增效劑通過(guò)多重機(jī)制增強(qiáng)抗真菌藥物的作用,尤其是其抑制HST3功能從而影響細(xì)胞壁完整性的機(jī)制有助于開(kāi)發(fā)新穎的抗真菌藥物或抗真菌藥物增效劑。
[Abstract]:Invasive fungal infection is still a clinically significant burden due to its high morbidity and high mortality, of which Candida albicans is the main pathogen of invasive fungal infection disease, such as Nostoglobinemia. However, the existing antifungal drugs have few kinds and many side effects, and the research and development speed of the antifungal medicine is not matched with the speed of the fungus resistance, and the novel antifungal medicine or the synergist of the existing antifungal medicine is more and more important. As a medicine for the clinical treatment of the rough skin disease, the bendamine (vitamin B3) has been proved to be an effective medicine for treating skin cancer and ataxia in recent years. In addition, in vitro experiments have been shown to be able to inhibit the growth of a plurality of fungi in vitro, and therefore, the present study is to explore whether or not the smoke and amine can be used as a synergist of an antifungal drug, with a view to reducing the burden on the clinical treatment of invasive fungal infections. The drug sensitivity test shows that the drug can effectively enhance the effect of the alkannin, the fluoroconrol, the amphotericin B and the capofene on the Candida albicans, and also can reduce the tailing phenomenon of the drug. The chess-type micro-liquid-based dilution method has been shown to be able to co-act with the above-mentioned drug-resistant Candida albicans, Candida albicans, Candida tropicalis, and Cryptococcus neoformans. The model of the infection of systemic candidiasis in mice was established. The results showed that the effect of flutamide on the infection of systemic candidiasis in mice was effectively enhanced, and the survival rate of mice was significantly improved. The results of the pathological section of the kidney of the mouse confirm that the combination of the phantamine with the fluconazole and the Capofene can also reduce the inflammatory reaction and the invasion of the kidney caused by the Candida albicans. The in vivo therapeutic effect of the combination of the tobacco and the Capofene showed a certain dose-dependence. In addition, the effect of amphotericin B on the treatment of systemic candidiasis is also enhanced to a certain extent. The immunoblotting method proves that the alkannin can induce the over-ethylation of the 56-position lysine on the histone H3 of the Candida albicans by inhibiting the action of the deacetylation-related gene HST3, Both amphotericin B and Capofene could not induce hyperethylation of 56, 9 and 18 lysine on H3. The flow cytometry (FCM) was used to find that a certain degree of apoptosis of Candida albicans can be induced by the amcinamide, and the cell cycle can be obviously changed, such that the G1 phase or the S phase is blocked, and these effects can help the anti-fungal drug to be anti-fungal. Using a laser confocal microscope, a transmission electron microscope, a flocculation test, a cell surface hydrophobic experiment and the like, we found that the content of mannose in the cell wall of the Candida albicans is reduced and the chitin content is increased by inhibiting the function of the HST3. (1, 3)-gluan exposure and cell wall relaxation, cell membrane shrinkage and edge blurry, cell organelle edge blurry, enhanced fungal flocculation capacity, increased cell surface hydrophobicity, and thus synergistic antifungal drug antifungal. In addition, the cell wall-related gene-deleted bacteria library was screened by a micro-liquid-based dilution method, and we found that the effect of the amantamine on the cell wall is closely related to the GIN4 gene. Further, we found that the amino acid metabolism of Candida albicans, the circulation of trivianic acid, the nitrogen metabolism, the glycolysis, the pentose phosphate pathway, and the like can be influenced by the tobacco chloroamine through the study of the metabolic group. in particular, the smoke and the amine can also lead to the increase of the content of the polyamine in the Candida albicans, This suggests that the ornithine deaminase or its coding gene in the pathway of the polyamine synthesis is likely to play an important role in the synergistic process of the amantamine. To sum up, the research of this subject confirms that the smoke and amine can be used as a synergist for effectively enhancing the treatment of the fungal infection of the invasive fungus, and the synergist is used for enhancing the action of the antifungal drug through the multiple mechanisms, in particular, that mechanism for inhibiting the function of the hst3 to affect the integrity of the cell wall is useful in the development of novel antifungal or antifungal drug potentiators.
【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(lèi)號(hào)】：R96
，
本文編號(hào)：2359869
[Abstract]:Invasive fungal infection is still a clinically significant burden due to its high morbidity and high mortality, of which Candida albicans is the main pathogen of invasive fungal infection disease, such as Nostoglobinemia. However, the existing antifungal drugs have few kinds and many side effects, and the research and development speed of the antifungal medicine is not matched with the speed of the fungus resistance, and the novel antifungal medicine or the synergist of the existing antifungal medicine is more and more important. As a medicine for the clinical treatment of the rough skin disease, the bendamine (vitamin B3) has been proved to be an effective medicine for treating skin cancer and ataxia in recent years. In addition, in vitro experiments have been shown to be able to inhibit the growth of a plurality of fungi in vitro, and therefore, the present study is to explore whether or not the smoke and amine can be used as a synergist of an antifungal drug, with a view to reducing the burden on the clinical treatment of invasive fungal infections. The drug sensitivity test shows that the drug can effectively enhance the effect of the alkannin, the fluoroconrol, the amphotericin B and the capofene on the Candida albicans, and also can reduce the tailing phenomenon of the drug. The chess-type micro-liquid-based dilution method has been shown to be able to co-act with the above-mentioned drug-resistant Candida albicans, Candida albicans, Candida tropicalis, and Cryptococcus neoformans. The model of the infection of systemic candidiasis in mice was established. The results showed that the effect of flutamide on the infection of systemic candidiasis in mice was effectively enhanced, and the survival rate of mice was significantly improved. The results of the pathological section of the kidney of the mouse confirm that the combination of the phantamine with the fluconazole and the Capofene can also reduce the inflammatory reaction and the invasion of the kidney caused by the Candida albicans. The in vivo therapeutic effect of the combination of the tobacco and the Capofene showed a certain dose-dependence. In addition, the effect of amphotericin B on the treatment of systemic candidiasis is also enhanced to a certain extent. The immunoblotting method proves that the alkannin can induce the over-ethylation of the 56-position lysine on the histone H3 of the Candida albicans by inhibiting the action of the deacetylation-related gene HST3, Both amphotericin B and Capofene could not induce hyperethylation of 56, 9 and 18 lysine on H3. The flow cytometry (FCM) was used to find that a certain degree of apoptosis of Candida albicans can be induced by the amcinamide, and the cell cycle can be obviously changed, such that the G1 phase or the S phase is blocked, and these effects can help the anti-fungal drug to be anti-fungal. Using a laser confocal microscope, a transmission electron microscope, a flocculation test, a cell surface hydrophobic experiment and the like, we found that the content of mannose in the cell wall of the Candida albicans is reduced and the chitin content is increased by inhibiting the function of the HST3. (1, 3)-gluan exposure and cell wall relaxation, cell membrane shrinkage and edge blurry, cell organelle edge blurry, enhanced fungal flocculation capacity, increased cell surface hydrophobicity, and thus synergistic antifungal drug antifungal. In addition, the cell wall-related gene-deleted bacteria library was screened by a micro-liquid-based dilution method, and we found that the effect of the amantamine on the cell wall is closely related to the GIN4 gene. Further, we found that the amino acid metabolism of Candida albicans, the circulation of trivianic acid, the nitrogen metabolism, the glycolysis, the pentose phosphate pathway, and the like can be influenced by the tobacco chloroamine through the study of the metabolic group. in particular, the smoke and the amine can also lead to the increase of the content of the polyamine in the Candida albicans, This suggests that the ornithine deaminase or its coding gene in the pathway of the polyamine synthesis is likely to play an important role in the synergistic process of the amantamine. To sum up, the research of this subject confirms that the smoke and amine can be used as a synergist for effectively enhancing the treatment of the fungal infection of the invasive fungus, and the synergist is used for enhancing the action of the antifungal drug through the multiple mechanisms, in particular, that mechanism for inhibiting the function of the hst3 to affect the integrity of the cell wall is useful in the development of novel antifungal or antifungal drug potentiators.
【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(lèi)號(hào)】：R96
，
本文編號(hào)：2359869
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