本文選題：阿薩希毛孢子菌 + 氟康唑�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景與目的阿薩希毛孢子菌(Tricrosporon asahii,T.asahii)是引起毛孢子菌感染(Trichosporonosis)的主要致病菌。近年來,隨著抗菌藥物的不規(guī)范使用、免疫抑制劑的廣泛使用以及艾滋病患者的增加,阿薩希毛孢子菌感染的發(fā)病率也越來越高。唑類藥物因其抗真菌譜廣、不良反應(yīng)小等優(yōu)勢被廣泛用于真菌感染,包括毛孢子菌感染的治療。但是,近幾年報道出了毛孢子菌對唑類藥物較高的MIC(Minimum inhibitory concentration)值,甚至是耐藥。ERG11基因編碼的羊毛甾醇14α-去甲基化酶為唑類抗真菌藥物的作用靶點,唑類藥物與其特異性結(jié)合后抑制該酶活性,從而阻遏真菌細(xì)胞膜重要成分——麥角甾醇的合成。既往已有研究顯示ERG11基因突變和或表達(dá)上調(diào)可引起白念珠菌等多種念珠菌以及新型隱球菌等真菌對唑類藥物耐藥。但尚未有關(guān)于ERG11基因表達(dá)在阿薩希毛孢子菌唑類耐藥中作用的研究報道。故本研究欲通過熒光定量PCR(Real-time PCR=q-PCR)技術(shù)檢測多對敏感菌株與耐藥菌株的ERG11基因的表達(dá)情況,探討ERG11基因在阿薩希毛孢子菌唑類耐藥中的作用以及基因表達(dá)量與藥物濃度的關(guān)系。為進(jìn)一步研究阿薩希毛孢子菌的耐藥機(jī)制及新藥靶點的開發(fā)奠定基礎(chǔ)。方法通過不斷增加氟康唑藥物濃度方法體外誘導(dǎo)獲得多株阿薩希毛孢子菌耐藥菌株,然后再將敏感菌株與耐藥菌株同時置于含有0~64μg/ml濃度氟康唑的酵母蛋白胨葡萄糖培養(yǎng)基(YPD)中培養(yǎng),提取RNA,使用q-PCR方法檢測所有受試菌株的ERG11基因的m RNA表達(dá)量。比較該基因在氟康唑敏感菌株與耐藥菌株中的表達(dá)差異,以及在不同濃度氟康唑作用下表達(dá)量的變化。并檢測分析氟康唑體外誘導(dǎo)過程中各菌株ERG11基因表達(dá)的變化。結(jié)果共獲得6株體外誘導(dǎo)成功的耐藥菌株。在無藥情況下,阿薩希毛孢子菌耐藥株組ERG11基因表達(dá)(7.542±5.311)高于敏感株組(1.014±0.012),兩組比較t=3.002,p=0.03,差異有統(tǒng)計學(xué)意義。在不同濃度氟康唑作用下,耐藥株組ERG11m RNA水平分別為9.183±3.226,13.657±5.428,15.292±7.007,13.720±8.550,13.949±2.960,13.123±6.429,亦高于敏感株組3.281±2.068,3.459±1.923,3.242±2.530,3.651±0.728,3.969±1.924,3.824±1.875,均p0.05,有統(tǒng)計學(xué)意義。但ERG11表達(dá)量與氟康唑濃度之間差異無顯著相關(guān)性(耐藥株rs=0.229,p=0.096;敏感株rs=0.166,p=0.357)。且在誘導(dǎo)耐藥過程中,雖然各受試菌株的ERG11表達(dá)量隨著其對氟康唑的耐藥性上升均呈現(xiàn)出上升趨勢,但只有2株菌株的耐藥性與其ERG11表達(dá)量之間有顯著等級相關(guān)性(BZP902:rs=0.865,p=0.003;BZP705:rs=0.847,p=0.002)。結(jié)論ERG11基因的表達(dá)水平與阿薩希毛孢子菌氟康唑耐藥有關(guān),但ERG11基因過表達(dá)是否會導(dǎo)致阿薩希毛孢子菌氟康唑耐藥,及其具體機(jī)制還有待于進(jìn)一步研究。
[Abstract]:Background & objective Tricrosporon asahiiae (T. asahii) is the main pathogen of Trichosporonosism.In recent years, with the irregular use of antimicrobial agents, the widespread use of immunosuppressants and the increase of AIDS patients, the incidence of Asaxia sporocystis infection is also increasing.Azoles are widely used in the treatment of fungal infections, including Trichosporum, due to their wide antifungal spectrum and small adverse reactions.However, in recent years, it has been reported that the higher value of MIC(Minimum inhibitory concentration of Cryosporium to azoles, and even the target of wool sterol 14 偽 -demethylase encoded by the resistance. ERG11 gene, as the target of antifungal agents of azoles, has been reported in recent years.The activity of the enzyme was inhibited by the specific binding of zolium, which inhibited the synthesis of ergosterol, an important component of fungal cell membrane.Previous studies have shown that mutation and up-regulation of ERG11 gene can induce resistance of Candida albicans and Cryptococcus neoformans.However, there have not been any studies on the role of ERG11 gene expression in the resistance of Asaxia sporidium to azotrazole.Therefore, the purpose of this study was to detect the expression of ERG11 gene in sensitive and resistant strains by fluorescence quantitative PCR(Real-time PCR q-PCR technique, and to explore the role of ERG11 gene in the resistance of Asaxia sporidium and the relationship between gene expression and drug concentration.It will lay a foundation for further study on the drug resistance mechanism of Asaxia and the development of new drug targets.Methods by increasing fluconazole concentration in vitro, many strains of Asaxia sporidium resistant strains were obtained.Then, the sensitive strains and the drug-resistant strains were cultured in yeast peptone glucose medium containing fluconazole (0 渭 g/ml) at the same time. The q-PCR method was used to detect the m RNA expression of ERG11 gene in all the tested strains.To compare the difference of expression of this gene between fluconazole-sensitive strain and drug-resistant strain, and the change of expression quantity under different concentration of fluconazole.The changes of ERG11 gene expression during fluconazole induction in vitro were detected and analyzed.Results A total of 6 drug-resistant strains were successfully induced in vitro.In the absence of drug, the expression of ERG11 gene was 7.542 鹵5.311 in the resistant strains of Asaxia and 1.014 鹵0.012 in the susceptible strains. The difference between the two groups was statistically significant.The levels of ERG11m RNA in the resistant strain group were 9.183 鹵3.226, 13.657 鹵5.428, 15.292 鹵7.007, 13.720 鹵8.550, 13.949 鹵2.960,949 鹵6.429, and 3.281 鹵2.0683.459 鹵1.9233.242 鹵2.530, 3.651 鹵0.728, 3.969 鹵1.9243.824 鹵1.875, respectively.However, there was no significant correlation between the expression of ERG11 and fluconazole concentration (0.229p0. 096 in resistant strain, 0.166 in susceptible strain and 0.357 in susceptible strain).In the course of inducing drug resistance, although the ERG11 expression of each tested strain showed an upward trend with the increase of fluconazole resistance, only two strains showed a significant grade correlation with their ERG11 expression, BZP902: Rs902: Rs902: Rs0.003 BZP705: rs0.847 p0.002.Conclusion the expression level of ERG11 gene is related to fluconazole resistance of Asaxia sporidium, but whether the overexpression of ERG11 gene leads to fluconazole resistance of Asaxia sporidium remains to be further studied.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R756

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