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目的:探讨白头翁汤正丁醇提取物butyl alcohol extract of Baitouweng decoction,BAEB对热带念珠菌(Candida tropicalis)毒力因子细胞表面疏水性、菌丝及生物膜形成的影响。方法:采用微量稀释法测定BAEB对热带念珠菌的最低抑菌浓度(MIC);XTT还原法测定BAEB对热带念珠菌生物膜抑制作用(SMIC80),Time-Kill法检测BAEB对热带念珠菌活菌数的动态影响;水-烃两相法测定BAEB对热带念珠菌细胞表面疏水性(cell surface hydrophobicity,CSH)的影响;荧光显微镜观察BAEB对热带念珠菌菌丝形态的影响;扫描电镜观察BAEB对热带念珠菌生物膜形态的影响;激光共聚焦显微镜检测BAEB对热带念珠菌生物膜荧光信号强度的影响;实时荧光定量PCR(q RT-PCR)检测菌丝及生物膜相关基因UME6,NRG1,ALST3的表达量变化。结果:BAEB对9株热带念珠菌的MIC在64~128 mg·L-1,对热带念珠菌生物膜的SMIC80为256~512 mg·L-1,FLZ对热带念珠菌的MIC在1~1 024 mg·L-1,SMIC80为1 024 mg·L-1或以上,Time-Kill曲线显示256,512 mg·L-1BAEB具有明显的杀菌作用,CSH实验显示128,256,512 mg·L-1BAEB均能够降低其表面疏水性,SEM观察到512 mg·L-1BAEB能够有效抑制热带念珠菌在硅胶导尿管上生物膜完整度,CLSM显示256,512 mg·L-1BAEB可以明显降低生物膜荧光信号强度,q RT-PCR检测显示在256,512 mg·L-1BAEB作用下,ALST3,UME6基因表达量显著下调,NRG1无明显变化。结论:BAEB可以抑制热带念珠菌细胞表面疏水性、菌丝及生物膜形成等毒力因子。
Objective: To investigate the effect of BAEB on the cell surface hydrophobicity, mycelium and biofilm formation of Candida tropicalis virulence factors. Methods: The minimal inhibitory concentration (MIC) of BAEB against Candida tropicalis was determined by micro-dilution method. The inhibition of BAEB against C. albicans biofilms was determined by XTT reduction method (SMIC80) The effect of BAEB on the cell surface hydrophobicity (CSH) of Candida tropicalis was observed by water-hydrocarbon two-phase method. The effect of BAEB on the morphology of C. tropicalis was observed by fluorescence microscopy. The effects of BAEB on the fluorescence signal intensity of Candida tropicalis biofilm were detected by laser scanning confocal microscopy. The mycelial and biofilm-related genes UME6, NRG1 and ALST3 were detected by real-time fluorescence quantitative PCR (q RT-PCR) Changes in the amount of expression. Results: The MICs of BAEB against Candida tropicalis were 64 ~ 128 mg · L-1 for SMIC80 and 256 ~ 512 mg · L-1 for SMC of Candida tropicalis biofilms, while MIC for FLZ against Candida tropicalis was between 1 and 1 024 mg · L-1, SMIC80 was 1 024 mg · L-1 or more, Time-Kill curve showed that 256,512 mg · L-1BAEB had obvious bactericidal activity. CSH experiments showed that 128,256,512 mg · L -1 BAEB could reduce the surface Hydrophobicity, SEM observation showed that 512 mg · L-1BAEB can effectively inhibit the biofilm integrity of C. albicans on silicone catheters. CLSM showed that 256,512 mg · L -1 BAEB can significantly reduce the biofilm fluorescence signal intensity. Q RT-PCR The results showed that under the action of 256, 512 mg · L-1BAEB, the expression of ALST3 and UME6 genes were significantly down-regulated while there was no significant change in NRG1. CONCLUSION: BAEB can inhibit virulence factors such as cell surface hydrophobicity, mycelium and biofilm formation in Candida tropicalis cells.