本文關(guān)鍵詞：白色念珠菌ERG11基因突變與其耐三唑類抗真菌藥物的關(guān)系　出處：《鄭州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

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【摘要】：背景近些年來,在真菌感染患者的臨床治療中免疫抑制劑、廣譜抗生素以及糖皮質(zhì)激素等的使用愈來愈多見,此外,諸如氣管插管、呼吸機(jī)等侵入性治療操作的使用頻率愈來愈高,這些都可能導(dǎo)致患者中肺部真菌感染幾率的增大。最多見的造成深部感染的真菌主要為念珠菌、青霉菌、曲霉菌、隱球菌、孢子絲菌、和毛霉菌等幾種。深部真菌感染的愈后差,病死率高,藥物應(yīng)用相對局限。因此,對深部真菌感染的致病菌的鑒定對臨床診斷、治療、愈后等有著很重要的影響,但深部真菌感染的菌種鑒定是臨床微生物實(shí)驗(yàn)室的一項(xiàng)難題。在傳統(tǒng)的真菌的分類鑒定中,常采用的方法均是基于真菌菌種的形態(tài)學(xué)、生理生化等特點(diǎn),并參考菌株的生理生化指標(biāo)進(jìn)行綜合鑒定和分析。采用這種分析鑒定方法的弊端在于真菌菌株的檢出及菌株的培養(yǎng)需過程需要保證特殊的培養(yǎng)環(huán)境和培養(yǎng)步驟;另外,由于實(shí)際培養(yǎng)中大部分的真菌菌株的生長相對緩慢,這給真菌菌種的鑒定工作造成了相當(dāng)?shù)睦щy。最終也就降低了針對深部真菌感染的致病菌進(jìn)行鑒定獲得的鑒定結(jié)果對于臨床治療的意義。近年來,得益于分子生物學(xué)的快速發(fā)展和進(jìn)步,利用分子生物學(xué)的方法能夠快速、準(zhǔn)確地對真菌菌種進(jìn)行鑒定,且該方法的應(yīng)用范圍正變得愈來愈廣泛,現(xiàn)已逐漸發(fā)展為真菌菌種鑒定的重要輔助手段之一。念珠菌(Candida)在自然界中的分布即為廣發(fā),通�？梢詫θ梭w的皮下組織、內(nèi)臟和黏膜等造成侵襲,并且是能夠引起目標(biāo)對象全身性感染的條件致病真菌。在患者的臨床診治過程中,發(fā)現(xiàn)的致病真菌基本包括了白色念珠菌(Canida albicans)、光滑念珠菌(Canida glabrata)、熱帶念珠菌(Canida tropicalis)、近平滑念珠菌(Canida parapsilosis)以及克柔念珠菌(Canida krusel)等5大類。這里面在真菌的臨床診治上的致病菌以白色念珠菌出現(xiàn)概率最高。白色念珠菌的Secreted Aspartyl Proteinases(SAP)多基因家族至少有九名成員(SAP1到SAP9),通過編碼分泌產(chǎn)出型天冬氨酸蛋白酶(Saps),而該蛋白酶能夠參與到白色念珠菌對組織的侵襲性;白色念珠菌的組織侵襲性被公認(rèn)為是白色念珠菌作為致病菌感染人體的重要致病因素。實(shí)驗(yàn)證實(shí),當(dāng)SAP6基因被敲除時,白色念珠菌的生存能力明顯下降,對宿主細(xì)胞的侵襲力明顯降低,對組織的損傷顯著減少;因此,白色念珠菌所獨(dú)有的功能性基因SAP基因可用于白色念珠菌菌種的鑒定工作。唑類抗真菌藥物因其療效好、抗菌譜廣、生物利用吸收好、安全性比較高等優(yōu)點(diǎn),廣泛應(yīng)用于真菌感染的預(yù)防和治療。但是,鑒于唑類抗真菌藥物已長期、廣泛應(yīng)用于真菌制冷中,使得耐藥菌株日漸多現(xiàn),這使得肺部真菌病的防治難度愈來愈高。白色念珠菌獲得對唑類抗真菌藥物的耐藥性的具體機(jī)制不一而足,其中較為多見的一種機(jī)制如下,即編碼唑類抗真菌藥物靶酶的ERG11基因發(fā)生突變或過度表達(dá),并最終引起白色念珠菌菌株的耐藥性的產(chǎn)生,最為多見。唑類抗真菌藥物的靶酶為由ERG11基因通過編碼產(chǎn)生的14α-去甲基化酶(CYP51)。若ERG11基因發(fā)生突變則會給Ergllp的氨基酸序列帶來改變,從而使的14α-去甲基化酶的空間結(jié)構(gòu)有了變化,致使酶分子與藥物分子之間不能結(jié)合或結(jié)合力變?nèi)?致使真菌獲得耐藥性。有研究發(fā)現(xiàn),白色念珠菌菌株獲得耐藥性是通過唑類藥物的靶酶的過量表達(dá)實(shí)現(xiàn)的,實(shí)驗(yàn)通過將耐藥菌株和敏感菌株比較,發(fā)現(xiàn)大部分靶酶的m RNA含量升高的菌株,它們的MIC值也有較明顯的增高。比較基因組學(xué)(comparative genomics)技術(shù)是將基因測序技術(shù)和基因組圖譜二者相結(jié)合,通過將實(shí)際檢測到的基因序列同目前已知的基因和基因組結(jié)構(gòu)進(jìn)行比較,從而對解基因的功能、基因的表達(dá)機(jī)制,乃至物種的進(jìn)化過程等作出科學(xué)推斷的技術(shù)學(xué)科。本研究通過對耐藥白色念珠菌ERG11基因測序,與已知的標(biāo)準(zhǔn)白色念珠菌ERG11基因的進(jìn)行比較,通過對比發(fā)現(xiàn)兩者的基因序列中存在基因多態(tài)性差異,力圖發(fā)掘出可能的與耐藥性相關(guān)的基因;比較基因組學(xué)的方法對于念珠菌耐藥機(jī)制研究的發(fā)展和進(jìn)步有著極其重要的應(yīng)用。此次研究以白色念珠菌的ERG11基因?yàn)閷ο笳归_,通過比較基因組學(xué)的技術(shù)方法,對ERG11基因突變同念珠菌耐藥性產(chǎn)生之間的相關(guān)性進(jìn)行探討。材料與方法菌株的收集時間范圍:2013年3月至2014年3月;菌株采集來源:鄭州大學(xué)第一附屬醫(yī)院呼吸內(nèi)二科住院部收治的肺部真菌感染患者中所采集到的符合標(biāo)準(zhǔn)的臨床菌株共57株。菌株的培養(yǎng):將臨床上收集到的真菌菌株標(biāo)本依時間先后順序在沙保弱培養(yǎng)基上接種,溫度條件維持在35℃,培養(yǎng)時間為24~48h;持續(xù)觀察,當(dāng)念珠菌生長顯現(xiàn)出酵母樣菌落時,從中挑取單個菌落,并在科瑪嘉顯色培養(yǎng)基上接種,培養(yǎng)溫度35℃看,培養(yǎng)時間為24h;菌落鑒定的判據(jù)為培養(yǎng)菌產(chǎn)生顏色,若認(rèn)為有必要則可使用由法國生物梅里埃公司生產(chǎn)的VITEK-32型全自動細(xì)菌分析系統(tǒng)中的YBC卡對念珠菌菌種進(jìn)行進(jìn)一步鑒定。(若鑒定發(fā)現(xiàn)為某菌株分離自同一患者相同部位,則將其排除)。應(yīng)用ATBFugus3試條對32株白色念珠菌進(jìn)行藥敏實(shí)驗(yàn),實(shí)驗(yàn)步驟嚴(yán)格按照標(biāo)準(zhǔn)操作規(guī)程進(jìn)行,在35℃的有氧環(huán)境中培養(yǎng)24個小時,最后判讀藥敏結(jié)果并進(jìn)行記錄。氟康唑、伏立康唑、伊曲康唑分別是所采用的3種抗真菌藥。選取白色念珠菌保守基因序列SAP6作為目的基因進(jìn)行檢測,并根據(jù)Gen Bank公布的白色念珠菌SAP6基因序列,應(yīng)用Primer5.進(jìn)行0軟件設(shè)計(jì)白色念珠菌SAP6基因的PCR引物,對目的基因擴(kuò)增,將PCR產(chǎn)物純化并進(jìn)行測序,將測序結(jié)果與SAP6基因?qū)Ρ?序列相同者認(rèn)為是白色念珠菌。用以對照的標(biāo)準(zhǔn)的白色念珠菌ERG11基因序列以Gen Bank的公布為準(zhǔn),通過Primer5.0軟件對白色念珠菌ERG11基因的PCR引物開展設(shè)計(jì),經(jīng)目的基因的擴(kuò)增后,此后可對PCR產(chǎn)物進(jìn)行純化操作,此后進(jìn)行基因的測序工作�；驕y序的結(jié)果在Blast分析軟件中進(jìn)行,該軟件導(dǎo)出的基因序列與標(biāo)準(zhǔn)得到白色念珠菌ERG11基因標(biāo)準(zhǔn)序列X13296進(jìn)行比對和分析,最終確定是否有基因突變位點(diǎn)的產(chǎn)生。結(jié)果1.收集到符合標(biāo)準(zhǔn)的念珠菌有57株,鑒定菌株種類以白色念珠菌為主,共32株,占56.1%;光滑念珠菌緊隨其后,為12株,占到了21.1%;熱帶念珠菌8株,占到了14.0%;克柔念珠菌則只有3株,占到了5.3%;其他念珠菌僅為2株,只占到3.5%。2.通過32株白色念珠菌進(jìn)行體外藥敏實(shí)驗(yàn)可以發(fā)現(xiàn):白色念珠菌共32株,其中有4株對氟康唑有耐藥性,共有7株對伊曲康唑有耐藥性,而僅有2株對伏立康唑有耐藥性。3.對32株白色念珠菌進(jìn)行SAP6基因擴(kuò)增并測序,所有白色念珠菌都檢測出了SAP6基因。4.經(jīng)過實(shí)驗(yàn)分析32株白色念珠菌的ERG11基因測序結(jié)果,并將其與標(biāo)準(zhǔn)的X13296序列進(jìn)行比對分析,結(jié)果發(fā)現(xiàn):堿基突變位點(diǎn)共有37處,其中錯義突變則共9處有發(fā)現(xiàn)。敏感菌株有3株出現(xiàn)錯義突變,9株耐藥菌株中存在1~5處錯義突變。此次試驗(yàn)發(fā)現(xiàn)了白色念珠菌ERG11基因錯義突變及氨基酸改變,其中敏感菌株的錯義突變有T945A、G1309A、A530C,與此相對應(yīng)的氨基酸突變分別為D116E、V437I、K128T,耐藥菌株則分別T495A、A530C、T541C、G622A、G979A、A945C、G1309A、G1496A、G1728T,對應(yīng)的氨基酸突變分別為D116E、K128T、Y132H、V159I、E226D、D278N、V437I、G450E、M527I。結(jié)論通過對白色念珠菌ERG11基因測序,發(fā)現(xiàn)耐三唑類抗真菌藥物的白色念珠菌多有ERG11基因突變,白色念珠菌ERG11基因突變導(dǎo)致D116E、V437I、K128T氨基酸改變,與白色念珠菌對三唑類抗真菌藥物耐藥性無關(guān);D226E、G450E、Y132H氨基酸改變與白色念珠菌對三唑類抗真菌藥物耐藥性有關(guān);V159I、D278N、M527I氨基酸改變與白色念珠菌對三唑類抗真菌藥物耐藥性關(guān)系需要進(jìn)一步研究。多位點(diǎn)錯義突變可產(chǎn)生協(xié)同作用,使得耐藥性增加或交叉耐藥。一些耐三唑類抗真菌藥物的白色念珠菌ERG11基因未發(fā)現(xiàn)有意義的氨基酸改變,考慮有其他機(jī)制參與其耐藥。
[Abstract]:Background in recent years, the fungal infection in the clinical treatment of immunosuppressant, broad-spectrum antibiotics and glucocorticoid use more and more, in addition, such as endotracheal intubation, ventilator use and invasive treatment and operation frequency is higher, which may lead to an increased risk of pulmonary fungal infection in patients. The most common fungi causing deep infection are Candida, Penicillium, Aspergillus, Cryptococcus, spores, and mucormycosis. The healing of deep fungal infection is poor, the mortality rate is high, and the drug application is relatively limited. Therefore, the identification of pathogenic bacteria for deep fungal infection has a very important impact on clinical diagnosis, treatment, recovery and so on, but the identification of deep fungal infection is a difficult problem in clinical microbiology laboratory. In traditional classification and identification of fungi, the methods commonly used are based on the morphological, physiological and biochemical characteristics of fungi, and refer to the physiological and biochemical indicators of strain for comprehensive identification and analysis. The drawbacks of this method for the analysis and identification of the fungal strains and strain detection is to develop the process needs to ensure that the special culture environment and culture step; in addition, due to the actual training in most of the fungi growth is relatively slow, which caused considerable difficulties for the identification of fungal strains. In the end, the results of identification of the pathogenic bacteria for deep fungal infection have also been reduced for clinical treatment. In recent years, thanks to the rapid development of molecular biology and progress, using molecular biology method can quickly and accurately identify the fungal species, the application scope and the method is becoming more and more widely, has gradually developed into one of the important means of identification of fungi. The distribution of Candida (Candida) in nature is GFA, which can invade subcutaneous tissue, viscera and mucous membrane of human body, and is a conditional pathogenic fungus that can cause systemic infection of target subjects. In the process of clinical diagnosis and treatment of patients, including the basic pathogenic fungi found in Candida albicans (Canida albicans), Candida glabrata, Candida tropicalis (Canida glabrata) (Canida tropicalis), c.parapsilosis (Canida parapsilosis) and Candida krusei (Canida krusel) and other 5 categories. The highest probability of Candida albicans is the pathogenic bacteria in the clinical diagnosis and treatment of fungi. Secreted Aspartyl Proteinases of Candida albicans (SAP) gene family with at least nine members (SAP1 to SAP9), secreted aspartic proteinase production by encoding (Saps), and the protease can be involved in the invasion of Candida albicans to the organization; organization of invasive Candida albicans is considered as Candida albicans the important pathogenic factors of human infection. Experiments show that when the SAP6 gene was knockout, Candida albicans viability decreased significantly, the host cell invasion significantly reduced tissue damage significantly reduced; therefore, the functional SAP genes unique to Candida albicans can be used for identification of Candida albicans strains. Azole antifungal drugs are widely used in the prevention and treatment of fungal infections due to their good efficacy, wide antimicrobial spectrum, good bioavailability and high safety. However, in view of the fact that azole antifungal drugs have been widely used in fungal refrigeration for a long time, the drug-resistant strains are becoming more and more frequent. This makes the prevention and treatment of pulmonary fungal diseases more and more difficult. This is not the only one specific mechanism of Candida albicans acquire resistance to azole antifungal drugs, one of the more common mechanisms are as follows, namely ERG11 gene encoding the target enzyme of azole antifungals mutated or overexpressed, and eventually lead to the resistance of Candida albicans strains, the most common. The target enzyme of the azolic antifungal agent is the 14 alpha demethylation enzyme (CYP51) produced by the encoding of the ERG11 gene. If ERG11 gene mutation occurs, it will change the amino acid sequence of Ergllp, resulting in the change of the spatial structure of the 14 alpha demethylation enzyme, resulting in the lack of binding or binding force between enzyme molecules and drug molecules, resulting in the drug resistance of fungi. It has been found that the resistance of Candida albicans isolates is achieved by over expression of target enzyme of azole drugs. Through comparing the resistant strains and sensitive strains, it is found that most of the m RNA strains with higher target enzymes have higher MIC values. Comparative genomics (comparative genomics) technology is the gene sequencing and genome combination of the two, compared with the known gene and genome structure through gene sequences will be detected, and the mechanism for the expression of the solution of gene function, gene technology, and evolution process to make scientific inference the. Through the study on drug resistance of Candida albicans ERG11 gene sequencing, compared with the standard of Candida albicans ERG11 gene known, by comparing the differences exist between genetic polymorphism of gene sequences, trying to explore the resistance associated gene; comparative genomics method has important applications for the study of mechanism of drug resistance of Candida albicans the development and progress of. In this study, the ERG11 basis of Candida albicans was launched, and the correlation between ERG11 gene mutation and Candida resistance was discussed through comparative genomics. The collection time and range of materials and methods were from March 2013 to March 2014. Strain collection sources: 57 strains of clinical isolates collected from two Department of respiratory medicine in the First Affiliated Hospital of Zhengzhou University were collected from the Department of pulmonary fungal infection in inpatient department. Culture of strains: the specimens collected from clinical fungi in chronological order
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R519

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