白色念珠菌氟康唑耐药相关基因的差异显示研究

Identification of Genes Related to Fluconazole Resistance in Candida albicans by Differential Display-PCR

采用差异显示PCR技术 (DifferentialDisplay PCR ,DD PCR)寻找白色念珠菌的氟康唑耐药相关基因。体外用含氟康唑的酵母培养基 (YEPD)诱导培养临床白色念珠菌氟康唑敏感株 4 35 (对咪康唑耐药 ) ,诱导 80d后得到氟康唑耐药子代 4 35 2 (MIC =1 2 8μg mL)。DD PCR比较 4 35 2、4 35分别在含氟康唑、不含药的YEPD液基中的基因表达 ,找到 3个明显差异片段 ,分别与数据库中白色念珠菌的醇脱氢酶基因ADH1、多药耐药基因CDR1及拓扑异构酶基因TOP2有高度同源性。半定量RT PCR中证实了ADH1、CDR1在氟康唑耐药株中的差异表达 ;对已知氟康唑耐药基因MDR1作半定量RT PCR时发现MDR1在氟康唑耐药株 4 35 2中无表达 ,而在氟康唑敏感株 4 35中有表达。结果表明 ,ADH1、CDR1基因的高表达与白色念珠菌氟康唑耐药性形成相关 ,ADH1可能是新的耐药基因 ;

Differential Display PCR technique(DD PCR)was employed to identify genes related to fluconazole resistance in Candida albicans . One fluconazole susceptible Candida albicans isolate 435(resistant to miconazole) from vagina was inducted to resist the drug by culturing in YEPD broth with increasing fluconazole concentration in vitro. The resistant isolate 435 2 (MIC =128μg/mL )appeared after being incubated for 80 days. There were three groups in this experiment: 435 2 cultured in YEPD broth with 64μg/mL fluconazole as Research group; 435 2 cultured in YEPD broth without fluconazole as Control group 1; 435 cultured in YEPD broth without fluconazole as Control group 2 Comparisons were done with DD PCR in gene expressions among the three groups. Three differentially displayed bands were found which showed high homogeneity to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1 genes of C.albicans respectively. The up regulated expression of ADH1 and CDR1 associated with fluconazole resistance was further confirmed by RT PCR. MDR1 that was known for its expression in fluconazole resistant isolate was now found down regulated in 435 2 cultured either in the presence or in the absence of fluconazole. These results indicated that the up regulated expression of ADH1 and CDR1 was associated with fluconazole resistance. ADH1 was possibly a novel fluconazole resistant gene. The expression of MDR1 may exist either in fluconazole susceptible isolate or in other azoles resistant isolates.