發(fā)布時間：2018-03-09 18:53

  本文選題：表觀遺傳學(xué)　切入點：白念珠菌　出處：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分曲古菌素A對唑類藥物體外抗白念珠菌活性增效作用的研究 第一章曲古菌素A對唑類藥物體外抗白念珠菌等常見病原真菌活性的影響 目的選擇敏感性不同的白念珠菌菌株、組蛋白去乙�；富蛉笔е昙捌渌Ｒ姴≡湍妇徒z狀真菌,研究曲古菌素A對氟康唑、伊曲康唑和伏立康唑抗白念珠菌等常見病原真菌的活性是否有增效作用。方法微量稀釋法檢測0.25μg/m1曲古菌素A與氟康唑、伊曲康唑和伏立康唑分別合用時對白念珠菌敏感株、耐藥株和常見病原真菌的最低抑菌濃度,并檢測白念珠菌的最小殺菌濃度,檢測組蛋白去乙�；妇幋a基因缺失株HDA1△和RPD3△對三種唑類藥物的體外敏感性。結(jié)果0.25μg/ml曲古菌素A與氟康唑、伊曲康唑和伏立康唑合用可不同程度地減少白念珠菌的拖尾現(xiàn)象,或表現(xiàn)為生長孔菌量減少,少數(shù)菌株的24h最低抑菌濃度降低,對最小殺菌濃度無明顯影響；曲古菌素A不影響其它常見病原酵母菌和絲狀真菌與唑類藥物的體外敏感性；與同源株比較,HDA1△和RPD3△,體外敏感性增加。結(jié)論曲古菌素A對唑類抗真菌藥物體外抗白念珠菌活性有增效作用,對其體外殺菌作用無影響。曲古菌素A與唑類抗真菌藥物合用對除白念珠菌外的常見病原酵母菌和絲狀真菌的體外敏感性無增效作用。組蛋白去乙�；妇幋a基因HDA1和RPD3可能與白念珠菌體外敏感性相關(guān)。 第二章曲古菌素A對唑類藥物體外增效作用的機(jī)制初探 目的研究白念珠菌組蛋白去乙�；妇幋a基因的表達(dá)水平,以及組蛋白去乙�；妇幋a基因和外排泵基因在體內(nèi)外獲得性耐藥形成過程中表達(dá)水平的變化。應(yīng)用羅丹明123(Rh123)檢測曲古菌素A對白念珠菌外排泵功能的影響。方法體外誘導(dǎo)白念珠菌對氟康唑耐藥,收集藥物作用前的白念珠菌和氟康唑環(huán)境下的體內(nèi)外獲得性耐藥的白念珠菌。Trizol法提取白念珠菌RNA,白念珠菌ACT1作為內(nèi)參基因,熒光定量RT-PCR檢測白念珠菌臨床株組蛋白去乙�；妇幋a基因HDA1、RPD3、 Hos1、HOS2、HOS3的表達(dá)水平,以及組蛋白去乙�；妇幋a基因和外排泵基因CDR1、CDR2、MDR1和FLU1在體內(nèi)外獲得性耐藥形成過程中表達(dá)水平的變化。應(yīng)用熒光染料Rh123處理白念珠菌,熒光分光光度計檢測白念珠菌臨床株、體外誘導(dǎo)耐藥菌株在曲古菌素A作用下的熒光變化,以檢驗白念珠菌外排泵功能的變化。結(jié)果藥物作用前,白念珠菌的組蛋白去乙�；妇幋a基因有不同程度表達(dá),與菌株自身敏感性和曲古菌素A對菌株的增效作用無關(guān)；體內(nèi)外獲得性耐藥形成過程中HDA1、RPD3、HOS1、HOS2和HOS3表達(dá)水平有不同程度增高,外排泵基因CDR1、CDR2、MDR1表達(dá)水平呈不同程度增加,LU1表達(dá)水平變化不明顯。曲古菌素A對白念珠菌臨床株短時間作用后,外排泵功能無明顯變化,氟康唑／曲古菌素A誘導(dǎo)組的Rh123熒光值高于氟康唑誘導(dǎo)組的Rh123熒光值。結(jié)論組蛋白去乙�；揎椏赡苁前啄钪榫馀疟没蛩阶兓纳嫌螜C(jī)制之一。 第二部分念珠菌對常見抗真菌藥物的體外敏感性測定 第一章微量稀釋法檢測念珠菌對抗真菌藥物敏感性的兩種觀察結(jié)果方法的比較 目的應(yīng)用微量稀釋法檢測念珠菌體外抗真菌藥物敏感性,比較肉眼觀察和體視顯微鏡觀察結(jié)果的一致性及優(yōu)缺點。方法參考CLSI的微量稀釋法M27-A3,檢測203株念珠菌對4種唑類藥物(咪康唑、酮康唑、益康唑、聯(lián)苯芐唑)及4種非唑類藥物(制霉菌素、利拉萘酯、萘替芬、特比萘芬)的體外敏感性,并同時應(yīng)用肉眼和體視顯微鏡兩種方法觀察結(jié)果。結(jié)果1.唑類藥物：咪康唑、酮康唑、益康唑和聯(lián)苯芐唑的一致性范圍為：87.68%～96.06%；2.非唑類藥物：制霉菌素、利拉萘酯、萘替芬、特比萘芬的一致性范圍為91.63%～99.51%。3.與肉眼觀察比較,體視顯微鏡觀察結(jié)果具有以下優(yōu)點：成像有立體感；可以觀察到肉眼看不見的微量菌生長；易于區(qū)分待測菌和污染菌生長；4.除利拉萘酯、萘替芬和聯(lián)苯芐唑外,咪康唑、酮康唑等均對念珠菌有良好的體外抑菌作用。結(jié)論對于微量稀釋法體外檢測念珠菌的抗真菌藥物敏感性,應(yīng)用體視顯微鏡可以輔助觀察結(jié)果,且與肉眼讀取結(jié)果有較好一致性,兩種方法結(jié)合有助于體外抗真菌藥物敏感性結(jié)果的判斷。 第二章盧立康唑及其它6種咪唑類抗真菌藥物對常見念珠菌的體外活性檢測 目的評價盧立康唑及其它6種咪唑類藥物對臨床分離常見念珠菌的體外敏感性。方法參考CLSI的微量稀釋法M27-A3方案,檢測5種共183株臨床分離念珠菌對盧立康唑、酮康唑、咪康唑、益康唑、克霉唑、舍他康唑、聯(lián)苯芐唑7種咪唑類藥物的體外敏感性。結(jié)果酮康唑、咪康唑、益康唑、克霉唑、舍他康唑和聯(lián)苯芐唑的體外最低抑菌濃度范圍(幾何均數(shù))分別為0.03-8(0.067)μg/ml、0.03-16(0.071)μg/ml、0.03-8(0.207)μg/ml、0.03-8(0.061)μg/ml、0.03-16(0.187)μg/ml.和0.03～16(1.050)μg/ml�？拐婢滤幈R立康唑?qū)?種念珠菌均有較好的體外敏感性,最低抑菌濃度范圍0.03～8μg/ml,幾何均數(shù)為0.087μg/ml, MIC50和MIC90分別為0.06μg/ml和0.5μg/ml。包括盧立康唑在內(nèi),各受試藥物均有部分菌株相對不敏感。結(jié)論除聯(lián)苯芐唑外,盧立康唑等其它6種咪唑類藥物均對念珠菌有良好的體外抗菌活性,但存在少數(shù)菌株相對不敏感。
[Abstract]:Study on the synergistic effect of triacycin A on the activity of anti Candida albicans
The effect of triacycin A on the activity of the common pathogenic fungi of Candida albicans against Candida albicans in the first chapter
Objective to select the different sensitivity of Candida albicans strains, histone deacetylase gene deletion strains and other pathogenic yeasts and filamentous fungi, study of trichostatin A on whether there is a synergistic effect of fluconazole, itraconazole and voriconazole against Candida albicans and other pathogenic fungi. The activity of A and fluconazole detection method of micro dilution method 0.25 g/m1 in archaea, itraconazole and voriconazole were combined to Candida albicans strains, the minimum inhibitory concentration of drug resistant strains and pathogenic fungi, and to detect the minimal bactericidal concentration of Candida albicans, detection of histone acetylation in vitro sensitivity encoding gene deletion strains HDA1 and RPD3 Delta Delta on three kinds of azole drugs. Results in A and fluconazole 0.25 g/ml Qugu bacteria, itraconazole and voriconazole combined reduced smearing of Candida albicans, or for growth of Kong Jun Reduce 24h, the minimum inhibitory concentration of a few strains were decreased and had no obvious effect on the minimum bactericidal concentration; trichostatin A does not affect the sensitivity of other common pathogenic yeasts and filamentous fungi with azole drugs in vitro; and homologous strains, HDA1 and delta RPD3 Delta, in vitro sensitivity increased. Conclusion trichostatin A had synergism effect of anti Candida albicans in vitro activity of azole antifungal drugs had no effect on the in vitro bactericidal action of trichostatin A and azole antifungal drug sensitivity of Candida albicans in the common pathogenic yeasts and filamentous fungi in vitro. No synergistic effect of histone deacetylase gene encoding HDA1 and RPD3 and in vitro susceptibility of Candida albicans.
A preliminary study on the mechanism of the synergistic effect of trazocin A on azolic drugs in the second chapter
Objective to study the expression of Candida albicans histone acetyltransferase encoding gene, expression and histone deacetylase gene encoding and efflux pump gene acquired resistance in vitro and in vivo formation process. The application of Luo Danming 123 (Rh123) to detect effects of trichostatin A on Candida albicans efflux pump function the method for in vitro induction of Candida albicans resistant to fluconazole, extraction of Candida albicans RNA Candida albicans.Trizol was collected before the drug effects of Candida albicans and fluconazole environment in vivo and acquired drug resistance of Candida albicans ACT1 as reference gene, fluorescence quantitative RT-PCR detection of clinical isolates of Candida albicans histone deacetylase gene encoding HDA1, RPD3, Hos1, HOS2, HOS3 expression, and histone deacetylase gene encoding and efflux pump genes CDR1, CDR2, MDR1 and FLU1 during the formation of acquired drug resistance in vitro and in vivo in table The change of the level of application. The fluorescent dye Rh123 treatment of Candida albicans, fluorescence spectrophotometer detection of clinical isolates of Candida albicans resistant strains in vitro, fluorescence changes in trichostatin A under the action of the change of pumping function to test the Candida albicans outside. The results of drug action, Candida albicans histone deacetylase genes encoding enzymes have different levels of expression, has nothing to do with the strain sensitivity and trichostatin A synergistic effect on the strains; in vivo acquired drug resistance during the formation of HDA1, RPD3, HOS1, HOS2 and HOS3 expression levels increased, efflux pump gene CDR1, CDR2, MDR1 expression level increased, the expression level of LU1 did not change significantly. Trichostatin A in Candida albicans clinical isolates after a short period of time, the function of efflux pump has no obvious change, Rh123 fluorescence / fluconazole induced by trichostatin A group was higher than that of fluconazole induced group Rh The 123 fluorescence value. Conclusion histone deacetylation modification may be one of the upstream mechanisms of the change in the gene level of Candida albicans efflux pump.
In vitro sensitivity of second Candida albicans to common antifungal agents
Comparison of two methods for detecting susceptibility to Candida albicans against fungal drugs in the first chapter
Antifungal susceptibility testing of Candida albicans in vitro by microdilution method, the consistency of the advantages and disadvantages of visual observation and stereological microscopy results. Microdilution reference method M27-A3 CLSI, detection of 203 strains of Candida to 4 azole drugs (miconazole, ketoconazole, miconazole, bifonazole) and 4 non azole drugs (nystatin, liranaflate, naftifine, terbinafine) in vitro sensitivity of two methods and application of visual and stereo microscope. Results 1. azole drugs: ketoconazole, miconazole, Econazole and bifonazole consistency range 87.68% ~ 96.06%; 2. non azole drugs: nystatin, liranaflate, naftifine, terbinafine consistency range is 91.63% ~ 99.51%.3. compared with the naked eye, microscope observation has the following advantages: imaging three-dimensional sense can be observed; The naked eye cannot see the growth of trace bacteria; bacteria and bacteria to distinguish pollution to be tested; 4. except liranaftate, butenafine and bifonazole, miconazole, ketoconazole, have good antibacterial effect in vitro of Candida albicans. Conclusion for antifungal drug sensitivity in vitro microdilution detection of Candida albicans the results can be used to observe, auxiliary stereo microscope, and with the naked eye to read the results have better consistency, two methods contribute to the in vitro antifungal susceptibility of the judgment.
In vitro activity detection of Candida common Candida in the second chapter of luopazolazole and other 6 imidazole antifungal agents
鐩殑璇勪環(huán)鍗㈢珛搴峰攽鍙?qiáng)鍏跺?

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