摘要目的 探讨粉防己碱抑制耐氟康唑热带念珠菌外排泵的机制.方法 通过MTT法确定24 h粉防己碱对耐氟康唑热带念珠菌的无细胞毒性剂量,并以此剂量处理5株耐氟康唑热带念珠菌.粉防己碱组(20 mg/L)及对照组(PBS)加入不同浓度的氟康唑(10~50 mg/L)处理48 h以测定MIC值.半定量PCR分析粉防己碱(20 mg/L)作用24 h前后,耐氟康唑热带念珠菌外排泵基因CDR1和MDR1转录水平的变化;流式细胞术检测粉防己碱(20 mg/L)作用24 h后对耐氟康唑热带念珠菌罗丹明123的蓄积能力的影响.结果 粉防己碱对耐氟康唑热带念珠菌的最大无细胞毒性剂量为20 mg/L.以此浓度处理5株耐氟康唑热带念珠菌,与对照组相比,MIC的范围由64(64~128)mg/L减少到32(24~64)mg/L(P<0.05).粉防己碱可以将耐氟康唑热带念珠菌CDR1和MDR1基因mRNA水平的表达由0.905±0.026及0.649±0.028分别下调至0.715±0.131和0.342±0.068(均P<0.05).粉防己碱作用后的耐氟康唑热带念珠菌对罗丹明123的蓄积量提升至(37.7±3.5)%,高于对照组的(16.3±2.2)%(P<0.05).结论 粉防己碱可以减少耐氟康唑热带念珠菌外排蛋白的表达,增加细胞内药物的蓄积,从而抑制氟康唑的耐药作用.

abstractsObjective To explore the mechanism underlying inhibitory effect of tetrandrine on the efflux pump in fluconazole-resistant Candida tropicalis.Methods The non-cytotoxic dose of tetrandrine to Cadida tropicalis at 24 h was determined by MTT method.Tetrandrine by the determined dose was applied to five strains of fluconazole-resistant Candida tropicalis.The tetrandrine-treated group(20 mg/L)and the control group(PBS)of Candida tropicalis were then added with various concentrations of fluconazole(10-50 mg/L) for 48 h to determine the minimal inhibitory concentration (MIC).Semi-quantitative reverse transcription-PCR was used to examine the relative transcriptional level shift of CDR1 and MDR1 genes in these fluconazole-resistant strains before and after treatment with 20 mg/L tetrandrine for 24 h.Flow-cytometry was used to determine the effect of tetrandrine on rhodamine 123 accumulation capacity in Candida tropicalis at 24 h.Results The maximum non-cytotoxic dose of tetrandrine to Candida tropicalis was 20 mg/L as determined by MTT assay.Compared with the control group,in the 5 strains of fluconazole-resistant Candida tropicalis treated with maximum non-cytotoxic dose,the MIC of fluconazole decreased from 64(range 64-128)mg/L to 32(range 24-64)mg/L(P<0.05).Reverse transcription-PCR showed that the mRNA expression levels of CDR1 and MDR1 genes was reduced from 0.905±0.026 and 0.649±0.028 to 0.715±0.131 and 0.342±0.068,respectively(all P<0.05).After treatment with tetrandrine,the rhodamine 123 accumulation of fluconazole-resistant Candida tropicalis was elevated to(37.7 ± 3.5)%,compared with (16.3 ± 2.2)% in the controls (P<0.05).Conclusion Tetrandrine may down-regulate the expression of efflux pump genes and increase intracellular drug accumulation in fluconazole-resistant Candida tropicalis,thereby inhibiting the fluconazole resistance in this fungus.