体外诱导近平滑、热带念珠菌对氟康唑、两性霉素B耐药分子机制研究

发布时间：2019-04-15 18:14

【摘要】：目的:检测近平滑念珠菌、热带念珠菌对FCZ、AMB的药物敏感性，体外诱导近平滑念珠菌、热带念珠菌的FCZ耐药株及AMB耐药株，并检测诱导的耐药株中耐药基因的表达情况，探讨其对FCZ、AMB耐药的分子机制。方法：先以MTT微量液基稀释法检测近平滑念珠菌、热带念珠菌对FCZ、AMB的药物敏感性，再用FCZ、AMB浓度递增法体外诱导其耐药株,并用实时荧光定量PCR法检测野生株与耐药株耐药基因的表达水平。结果：近平滑念珠菌、热带念珠菌的野生株有CYP51A、CYP51B、CDR1、CDR2、MDR1、MDR2、Yap1、Sho1mRNA低水平表达，，而近平滑念珠菌、热带念珠菌的AMB耐药株中CYP51A、CYP51B、CDR1、CDR2、MDR1、MDR2、Yap1、Sho1基因的相对表达量明显上升，差异有统计学意义（P＜0.05）；近平滑念珠菌、热带念珠菌的FCZ耐药株中CYP51A、CDR1、CDR2、MDR1、Yap1、Sho1基因的相对表达量明显高于其野生株，差异有统计学意义（P＜0.05），而CYP51B、MDR2基因的表达量在近平滑念珠菌、热带念珠菌FCZ耐药株诱导形成过程中无明显变化（P0.05）。结论：CYP51A、CYP51B、CDR1、CDR2、MDR1、MDR2、Yap1、Sho1基因过度表达与近平滑念珠菌、热带念珠菌对AMB耐药有关；CYP51A、MDR1、CDR1、CDR2、Yap1、Shol基因的过表达与近平滑念珠菌、热带念珠菌对FCZ耐药有关，而近平滑念珠菌、热带念珠菌FCZ耐药株诱导形成过程中CYP51B、MDR2基因的表达量无明显变化。近平滑念珠菌、热带念珠菌长期接触FCZ、AMB后可诱导麦角固醇合成过程中的关键酶基因、外排泵基因及信号传导相关基因过表达，从而产生耐药性。
[Abstract]:Objective: to detect the drug sensitivity of Candida albicans and Candida tropicalis to FCZ,AMB, to induce Candida meliformis, FCZ-resistant strains and AMB-resistant strains of Candida tropicalis in vitro, and to detect the expression of drug-resistant genes in the drug-resistant strains induced by Candida tropicalis and Candida tropicalis. To investigate the molecular mechanism of its resistance to FCZ,AMB. Methods: MTT microdilution method was used to detect the drug sensitivity of Candida solanum and Candida tropicalis to FCZ,AMB, and then the drug-resistant strains were induced by increasing the concentration of FCZ,AMB in vitro. Real-time fluorescence quantitative PCR was used to detect the expression level of drug-resistant genes in wild and drug-resistant strains. Results: low level expression of CYP51A,CYP51B,CDR1,CDR2,MDR1,MDR2,Yap1,Sho1mRNA was found in wild strains of Candida tropicalis and Candida tropicalis, while CYP51A,CYP51B,CDR1,CDR2,MDR1,MDR2,Yap1, was found in AMB resistant strains of Candida solani and Candida tropicalis. The relative expression of Sho1 gene increased significantly (P < 0.05). The relative expression of CYP51A,CDR1,CDR2,MDR1,Yap1,Sho1 gene in FCZ resistant strains of Candida leiomycetes and Candida tropicalis was significantly higher than that of wild strains (P < 0.05), while CYP51B, was significantly higher than that of wild strains (P < 0.05). The expression of MDR2 gene did not change significantly during the induction of drug-resistant strains of Candida moniliforme and Candida tropicalis FCZ (P0.05). Conclusion: the overexpression of CYP51A,CYP51B,CDR1,CDR2,MDR1,MDR2,Yap1,Sho1 gene is related to the drug resistance of Candida solanum and Candida tropicalis to AMB. The overexpression of CYP51A,MDR1,CDR1,CDR2,Yap1,Shol gene was related to the resistance to FCZ of Candida solanum and Candida tropicalis, but the expression of CYP51B,MDR2 gene did not change during the induction of drug resistance of Candida tropicalis and Candida tropicalis FCZ. Long-term exposure to FCZ,AMB can induce the over-expression of key enzyme genes, efflux pump genes and signal transduction-related genes in ergosterol synthesis in Candida leiomycetes and Candida tropicalis, resulting in drug resistance.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R519.3

【参考文献】