本文選題：非白色念珠菌 + 藥物聯(lián)合��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：背景：近年來,非白色念珠菌由于其較高的臨床分離率和對(duì)氟康唑具有較高的獲得耐藥性或固有耐藥性,越來越引起人們的關(guān)注和重視。在臨床常見非白色念珠菌中,光滑念珠菌是第二位人類最常見的病原菌,具有較高的獲得耐藥性；克柔念珠菌雖然檢出率不高,但對(duì)氟康唑天然耐藥。它們較高的耐藥性給臨床成功治療非白色念珠菌感染帶來了挑戰(zhàn)。綜合考慮臨床常見非白色念珠菌的檢出率和耐藥性,本課題選擇光滑念珠菌和克柔念珠菌作為研究對(duì)象�？拐婢幬锲贩N有限,而開發(fā)一種新的抗真菌藥物耗時(shí)耗資巨大。因此,為應(yīng)對(duì)真菌不斷增加的耐藥性,越來越多的研究者關(guān)注于抗真菌藥物和非抗真菌藥物的聯(lián)合治療。他克莫司經(jīng)常用于器官移植患者的免疫抑制治療,而這些病人更容易遭受真菌感染。前期研究表明氟康唑和他克莫司聯(lián)用對(duì)耐藥的白色念珠菌有強(qiáng)大的協(xié)同作用,二者聯(lián)用對(duì)光滑念珠菌和克柔念珠菌是否也有協(xié)同作用值得研究。 目的：本研究旨在評(píng)價(jià)氟康唑和他克莫司聯(lián)合對(duì)不同敏感性的光滑念珠菌和克柔念珠菌的體外靜態(tài)和動(dòng)態(tài)抗真菌作用,并探索其協(xié)同作用可能的機(jī)制。 方法：分別采用棋盤法和時(shí)間-殺菌曲線法研究氟康畔和他克莫司聯(lián)用對(duì)不同敏感性的光滑念珠菌和克柔念珠菌的體外靜態(tài)和動(dòng)態(tài)抗真菌作用。根據(jù)M27-A3抗真菌藥物敏感性測(cè)定方案,以XTT法測(cè)定兩藥聯(lián)合作用24h和48h時(shí)對(duì)不同敏感性分離株的MIC值,以部分抑菌濃度指數(shù)(FICI)評(píng)價(jià)兩藥聯(lián)合作用效果；對(duì)于動(dòng)態(tài)聯(lián)合作用評(píng)價(jià),分別在藥物干預(yù)后的0,6,12,24和48h采用菌落計(jì)數(shù)法進(jìn)行菌落計(jì)數(shù),然后用藥物干預(yù)時(shí)間作橫坐標(biāo)、每毫升菌液菌落數(shù)的常用對(duì)數(shù)值作縱坐標(biāo)描繪不同藥物處理后的時(shí)間-殺菌曲線。藥物聯(lián)合抗真菌作用機(jī)制研究包括：加入鈣通道抑制劑貝尼地平,以平板劃線法直觀評(píng)價(jià)抑制鈣通道對(duì)兩藥聯(lián)合抗真菌作用的影響；以實(shí)時(shí)定量PCR法測(cè)定氟康唑耐藥基因ERG11, CDR1, PDH1和SNQ2在不同藥物干預(yù)后的相對(duì)表達(dá)量；用羅丹明6G作熒光染色劑,以流式細(xì)胞術(shù)評(píng)價(jià)他克莫司對(duì)真菌泵出氟康唑的影響。 結(jié)果：他克莫司增加了劑量依賴性敏感(MIC=32μg/mL)以及耐藥的光滑念珠菌和全部克柔念珠菌對(duì)氟康唑的敏感性,但菌株的敏感性不同,MIC值降低的程度也不同。對(duì)于MIC值大于等于256μg/mL的光滑念珠菌和克柔念珠菌菌株,他克莫司的加入使得氟康唑的MIC值降為原來的1/16,FICI值小于0.1；對(duì)于MIC值為32μg/mL的光滑念珠菌和克柔念珠菌菌株,他克莫司的加入使氟康唑的MIC值變?yōu)樵瓉淼?/4,FICI值約等于0.25；然而對(duì)于MIC值為8μg/mL的光滑念珠菌菌株,他克莫司的加入并沒有降低氟康唑的MIC值。動(dòng)態(tài)抗真菌作用實(shí)驗(yàn)結(jié)果進(jìn)一步證明了兩藥的協(xié)同作用：與氟康唑單用組相比,兩藥聯(lián)合對(duì)光滑念珠菌和克柔念珠菌在24h時(shí)每毫升菌液CFU的常用對(duì)數(shù)值分別下降了2.31和2.25個(gè)單位。另外,以平板劃線法觀察菌株在藥物作用后的生長(zhǎng)情況,結(jié)果表明貝尼地平的加入增加了兩藥聯(lián)合抗光滑念珠菌和克柔念珠菌的作用。對(duì)光滑念珠菌進(jìn)一步的機(jī)制研究表明氟康唑和他克莫司聯(lián)用顯著降低了ERG11和SNQ2基因的表達(dá)水平(表達(dá)量分別減少了90%和66%),顯著增加了CDRI基因的表達(dá),對(duì)PDH1基因的表達(dá)無顯著影響。流式細(xì)胞術(shù)的分析結(jié)果表明他克莫司抑制了氟康唑的外排,特別是在加入他克莫司作用40分鐘后抑制作用最明顯(胞內(nèi)熒光強(qiáng)度是對(duì)照組的2.76倍)。 結(jié)論：氟康唑和他克莫司聯(lián)用對(duì)光滑念珠菌和克柔念珠菌展現(xiàn)出協(xié)同作用。抑制鈣離子通道能夠增強(qiáng)兩藥協(xié)同抗真菌作用,說明兩藥協(xié)同抗真菌作用與鈣調(diào)節(jié)有關(guān)；兩藥協(xié)同抗光滑念珠菌的作用機(jī)制與減少了ERG11和SNQ2基因的表達(dá)和抑制了氟康唑的外排有關(guān)。他克莫司和氟康唑聯(lián)合用藥可能是克服光滑念珠菌和克柔念珠菌對(duì)氟康唑耐藥性的方法,本課題的研究給抗真菌藥物的研發(fā)提供了思路。
[Abstract]:Background : In recent years , non - Candida albicans has attracted more and more attention due to its high clinical separation rate and its high resistance to drug resistance or inherent drug resistance . In clinical non - candida albicans , Candida glabrata is the most common pathogenic bacterium in the second place , and has high resistance to resistance ;
Although the detection rate of Candida albicans is not high , it presents a challenge to the successful treatment of non - candida albicans infection . It has been considered that the combination therapy of antifungal drugs and non - antifungal drugs has been paid more and more attention to the detection rate and drug resistance of non - candida albicans .

Objective : To evaluate the in vitro static and dynamic antifungal effects of Fluconazole and Fluconazole in vitro static and dynamic antifungal activity against Candida albicans and Candida albicans , and to explore possible mechanisms for its synergistic action .

Methods : In vitro static and dynamic antifungal effects of Fluconazole and Kamox on the static and dynamic antifungal activity of Candida albicans and Candida albicans were studied by checkerboard method and time - sterilization curve method . The MIC values of different sensitive isolates were determined by XTT method at 24 h and 48 h .
For the evaluation of dynamic combined action , the colony counting method was used to count the colony count by colony counting method at 0 , 6 , 12 , 24 and 48 hours after drug intervention , then the time - sterilization curve after the treatment of different drugs was plotted with the drug intervention time as the abscissa and the logarithm of the colony count per milliliter .
Real - time quantitative PCR method was used to determine the relative expression of ER1 , CDR1 , PDH1 and SNQ2 of Fluconazole in different drug interventions .
Rodamin 6G was used as a fluorescent stain to evaluate the effect of fluconazol on fungal pump by flow cytometry .

RESULTS : The sensitivity of the dose - dependent sensitivity ( MIC = 32渭g / mL ) and the resistance of Candida glabrata and Candida albicans to fluconazol were increased . The sensitivity of the strains was different , and the MIC values were decreased . The MIC values of fluconazol were reduced to 1 / 16 and the FICI value was less than 0.1 for the strains with MIC values greater than or equal to 256 渭g / mL .
For Candida and Candida strains with MIC values of 32 渭g / mL , the MIC values of fluconazol were changed to 1 / 4 with a FICI value of approximately 0.25 ;
The results of flow cytometry showed that the combination of fluconazol and Kamox increased the expression level of the two drugs ( 90 % and 2.25 units , respectively ) . The results showed that the combination of fluconazol and Kamox increased the expression level of the two drugs ( 90 % and 66 % , respectively ) .

Conclusion : The combination of Fluconazole and Fluconazole showed a synergistic effect on Candida albicans and Candida albicans . The inhibition of calcium channel could enhance the synergistic antifungal effect of two drugs , suggesting that the synergistic antifungal effect of two drugs was related to calcium regulation .
The action mechanism of the two drugs in combination with anti - smooth candida is related to the reduction of the expression of the ER1 and SNQ2 genes and the inhibition of the efflux of fluconazol . The combination of tamostat and fluconazol can be a method for overcoming the resistance of Candida albicans and Candida albicans to fluconazol , and the research of the subject provides a thought for the development of antifungal drugs .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R969

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 于金龍;李妍;郭瓊杰;孫淑娟;;三唑類藥物與環(huán)孢菌素A的體外聯(lián)合抗真菌作用[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年08期

2 于金龍;孫淑娟;;酵母菌體外藥物敏感性試驗(yàn)方法研究進(jìn)展[J];齊魯藥事;2007年04期

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