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目的探讨氨溴索对体外白念珠菌(Candida albicans)生物被膜(biofilm,BF)结构及其真菌死亡率的影响。方法置片法培养90min、24h和48h的白念珠菌ATCC 10231 BF,分别用FITC-ConA染色后荧光显微镜下观察无氨溴索干预时BF的形成情况;激光共聚焦显微镜(CLSM)结合BF图像结构分析软件(Image Structure Analyzer,ISA)对BF结构参数进行定量分析,并采用Image Pro Plus 6.0分析软件分析不同浓度氨溴索干预下BF内真菌的死亡率。结果在无氨溴索干预下,运用荧光显微镜可观察到生长在玻片上呈绿色点片状的BF,且随着时间的延长绿色荧光增多增强。ISA软件定量分析显示:在空白对照组中,随着培养时间的延长,BF逐渐变厚,平均扩散距离(Average Diffusion Distance,ADD)和结构熵(Textual Entropy,TE)呈增加的趋势,区域孔率(Areal Porosity,AP)呈下降趋势。0.25mg/mL和1.5mg/mL氨溴索对90min BF、24h BF和48h BF分别作用8h后,与空白对照组相比,BF厚度、ADD、TE均减少(P<0.05);AP增加(P<0.05)。IPP软件分析显示:0.25mg/mL和1.5mg/mL氨溴索对90min BF、24h BF和48h BF分别作用8h后,与空白对照组相比,BF内真菌死亡率增加(P<0.05);高浓度组(1.5mg/mL)与低浓度组(0.25mg/mL)相比,高浓度组BF内真菌死亡率亦增加(P<0.05)。结论氨溴索干预各个时间段白色念株菌BF后,BF空间结构破坏和BF内死菌比例增加。且在一定浓度范围内,高浓度氨溴索作用BF后,其真菌死亡比例增加更明显。
Objective To investigate the effect of ambroxol on the structure and fungal mortality of Candida albicans biofilm (BF) in vitro. Methods The Candida albicans ATCC 10231 BF was cultured at 90, 24 and 48 h by Plating method. The formation of BF was observed by FITC-ConA staining and fluorescence microscopy. The confocal laser scanning microscopy (CLSM) The structure parameters of BF were quantitatively analyzed by Image Structure Analyzer (ISA), and the mortality of fungi in BF was analyzed by Image Pro Plus 6.0 analysis software with different concentrations of ambroxol. Results In the absence of ambroxol, EGF was observed on the glass slide by fluorescence microscopy, and the green fluorescence increased with time. The quantitative analysis of ISA software showed that, in the blank control group, BF gradually became thicker with the increase of incubation time, Average Diffusion Distance (ADD) and Textual Entropy (TE) Rate (Areal Porosity, AP) showed a downward trend. Compared with blank control group, the thickness of BF, ADD and TE decreased (P <0.05) at 0.25 mg / mL and 1.5 mg / mL ambroxol for 8 h after 90 min BF, 24 h BF and 48 h BF, respectively P <0.05). IPP analysis showed that the fungal mortality rate in BF was increased (P <0.05) compared with the blank control group after 0.25 mg / mL and 1.5 mg / mL ambroxol were respectively treated with 90 min BF, 24 h BF and 48 h BF for 8 h. The fungal mortality rate was also increased in the high-concentration group (P <0.05) compared with the low-concentration group (0.25 mg / mL) in the high-concentration group (1.5 mg / mL) Conclusions Ambroxol can inhibit the destruction of BF space structure and increase the proportion of dead bacteria in BF after the intervention of BF in different time periods. And in a certain concentration range, the high concentration of ambroxol after BF, the proportion of fungal death increased more significantly.