VVC患者临床分离白念珠菌Mrr2、CDR1与氟康唑耐药关系的研究

发布时间：2018-05-28 09:25

  本文选题：白念珠菌 + Mrr2　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:1、了解本地VVC患者念珠菌感染的菌种分布情况及其对FCA的敏感性;2、探讨VVC患者临床分离白念珠菌Mrr2突变与FCA耐药的关系;3、探讨VVC患者临床分离白念珠菌Mrr2、CDR1的mRNA表达水平与FCA耐药的关系,并进一步分析Mrr2与CDR1表达水平在FCA耐药中的相互调控作用;4、探讨VVC患者临床分离白念珠菌Mrr2基因突变与Mrr2的mRNA表达水平是否对FCA耐药具有相互作用。方法:采集2015年11月至2016年5月山西医科大学第二医院皮肤性病科就诊的可疑VVC患者阴道分泌物标本155份,并经过真菌镜检、CHROMagar念珠菌显色培养基及API 20C AUX念珠菌鉴定系统进行菌种鉴定,获得102株念珠菌。对其中的80株白念珠菌应用M27-A3微量肉汤稀释方法进行FCA药物敏感性试验。对80株白念珠菌分别提取DNA、聚合酶链反应(Polymerase Chain Reaction,PCR)扩增Mrr2基因片段、测序,寻找突变位点。对白念珠菌FCA耐药组和敏感组菌株分别提取总RNA,逆转录合成cDNA,实时荧光定量PCR(Real-time Fluorescence Quantitative PCR,FQ-RT-PCR)检测Mrr2、CDR1的mRNA表达量,并做相关统计学分析。结果:1、从155份阴道分泌物标本中,共获得念珠菌菌株102株。其中白念珠菌80株(78.4%),光滑念珠菌15株(14.7%),热带念珠菌4株(3.9%),近平滑念珠菌2株(2.0%),克柔念珠菌1株(1.0%)。2、对80株白念珠菌接种活化,并使用M27-A3微量肉汤稀释法进行FCA药物敏感性试验,结果显示:80株白念珠菌中有40株对FCA敏感,10株对FCA呈剂量依赖性敏感,30株对FCA耐药,耐药率为37.5%。3、对80株白念珠菌的Mrr2基因进行测序,其中FCA耐药组Mrr2基因突变率为56.67%;FCA敏感组Mrr2基因突变率为26.08%。对白念珠菌Mrr2基因突变与FCA耐药的关系采用四格表卡方检验,得出P值0.05,差异具有统计学意义。耐药组Mrr2基因突变率高于敏感组Mrr2的突变率。4、检测白念珠菌FCA敏感组和耐药组的Mrr2及CDR1的mRNA表达水平,经统计学分析:(1)白念珠菌FCA耐药组Mrr2的mRNA表达水平高于敏感组Mrr2的mRNA表达水平(1.10±0.50 VS 0.52±0.45),P0.05,差异具有统计学意义。(2)白念珠菌FCA耐药组CDR1的mRNA表达水平高于敏感组CDR1的mRNA表达水平(0.42±0.294 VS 0.25±0.289),P0.05,差异具有统计学意义。(3)CDR1与Mrr2呈正相关,r=37.6%。(4)Mrr2突变与Mrr2表达的交互作用分析,发现Mrr2基因发生突变且高表达组的耐药性是基因未发生突变且基因低表达组的耐药性的47.50倍,说明Mrr2基因突变与高表达对FCA耐药性的产生存在协同作用。结论:1、本地区VVC患者最主要的致病菌种是白念珠菌,对FCA耐药率较高,临床治疗VVC应依据药敏试验结果,选用敏感抗真菌药物进行精准治疗。2、VVC患者临床分离白念珠菌FCA耐药性可能与Mrr2突变有关。3、CDR1高表达可能直接导致白念珠菌对FCA耐药;Mrr2高表达可能直接导致白念珠菌对FCA耐药,且Mrr2高表达还可促进CDR1高表达进而介导白念珠菌对FCA耐药。4、Mrr2基因突变与高表达对FCA耐药性的产生可能存在协同作用。
[Abstract]:Objective to investigate the distribution of Candida albicans in local VVC patients and their sensitivity to FCA. To explore the relationship between Mrr2 mutation of clinical isolates of Candida albicans and drug resistance to FCA in patients with VVC. The relationship between the expression of mRNA and the drug resistance of FCA. Furthermore, the mutual regulation of Mrr2 and CDR1 expression level in FCA resistance was analyzed. The relationship between Mrr2 gene mutation and Mrr2 mRNA expression level in VVC patients was studied to determine whether the Mrr2 gene mutation and Mrr2 mRNA expression level had interaction with FCA resistance. Methods: from November 2015 to May 2016, 155 specimens of vaginal secretions from suspected VVC patients in Department of Dermatology and venereal Diseases, second Hospital of Shanxi Medical University, were collected. The strain of Candida albicans was identified by API 20C AUX system and the culture medium of CHROMagar was used to identify the species of Candida albicans, and 102 strains of Candida were obtained. The susceptibility of 80 strains of Candida albicans to FCA was tested by M27-A3 broth dilution method. DNA was extracted from 80 strains of Candida albicans. The Mrr2 gene fragment was amplified by polymerase chain reaction (PCR) and sequenced to find the mutation site. The total RNAs were extracted from Candida albicans FCA resistant and susceptible strains respectively, and the cDNAs were synthesized by reverse transcription. The mRNA expression of MRR2 CDR1 was detected by real-time quantitative PCR(Real-time Fluorescence Quantitative PCR FQ-RT-PCR1, and the correlation statistical analysis was made. Results 102 strains of Candida were obtained from 155 vaginal secretions. Among them, 80 strains of Candida albicans were inoculated and activated, 15 strains of Candida smooth were inoculated and activated, 4 strains of Candida tropicalis were found to be 3. 9%, 2 strains were close to Candida smoothing, 2 strains were close to Candida smoothing, 1 strain was 1 strain of Candida korgii, and inoculated and activated to 80 strains of Candida albicans, and the sensitivity of FCA was tested by M27-A3 broth dilution method. The results showed that 40 of the 80 strains of Candida albicans were sensitive to FCA. 10 strains were dose-dependent sensitive to FCA. 30 strains were resistant to FCA. The drug resistance rate was 37.5%. The Mrr2 gene of 80 strains of Candida albicans was sequenced. The mutation rate of Mrr2 gene in FCA resistant group was 56.67 and 26.08 in FCA-sensitive group. The relationship between Mrr2 gene mutation of Candida albicans and drug resistance of FCA was tested by four-grid table-chi-square test, P value was 0.05, the difference was statistically significant. The mutation rate of Mrr2 gene in resistant group was higher than that in sensitive group. The mRNA expression of Mrr2 and CDR1 in susceptible and resistant group of Candida albicans FCA was detected. The mRNA expression level of Mrr2 in Candida albicans FCA resistant group was 1.10 卤0.50 vs 0.52 卤0.45 P 0.05, the difference was statistically significant. 2) the CDR1 mRNA expression level of Candida albicans FCA resistant group was higher than that of sensitive CDR1 group. The expression level was 0.42 卤0.294 vs 0.25 卤0.289 P0.05, the difference was statistically significant. There was a positive correlation between CDR1 and Mrr2. There was a positive correlation between the mutation of MRR2 and the expression of Mrr2. It was found that the drug resistance of Mrr2 gene mutation and high expression group was 47.50 times higher than that of non-mutation gene and low gene expression group, which indicated that there was a synergistic effect of Mrr2 gene mutation and high expression on the drug resistance of FCA. Conclusion the main pathogen of VVC patients in this area is Candida albicans, and the rate of drug resistance to FCA is high. The clinical treatment of VVC should be based on the results of drug sensitivity test. The drug resistance of Candida albicans isolated from clinical isolates of Candida albicans FCA may be related to the mutation of Mrr2. The high expression of CDR1 may directly lead to the high expression of MRR2 in Candida albicans resistant to FCA, which may lead to the resistance of Candida albicans to FCA, and the drug resistance of Candida albicans to FCA may be directly related to the drug resistance of Candida albicans to FCA. Moreover, the high expression of Mrr2 can promote the high expression of CDR1 and mediate the mutation of MRR2 gene in Candida albicans resistant to FCA and the production of drug resistance to FCA by high expression.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;R711.3

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