脉冲磁场处理对格氏李斯特菌钙离子跨膜行为的影响

Effect of Pulsed Magnetic Fields on Ca^(2+) Transmembrane Behaviors of L. grayi

研究了脉冲磁场处理后格氏李斯特菌细胞Ca^(2+)的跨膜行为,包括对该探针进行荧光光谱特性以及细胞负载研究,建立了格氏李斯特菌胞内钙离子荧光强度测定方法,同时采用激光共聚焦荧光显微镜检测不同脉冲数下受脉冲磁场处理的后格氏李斯特菌胞内钙离子荧光强度和浓度的变化。研究结果表明,Fluo-4/AM荧光探针自身的荧光强度较小,能成功负载于格氏李斯特菌中,可用于格氏李斯特菌胞内游离Ca^(2+)浓度的测定,且最优负载条件是负载浓度6μmol/L,负载时间60 min,负载温度37℃。经不同脉冲数的脉冲磁场处理后,格氏李斯特菌胞内Ca^(2+)浓度与菌落总数的变化呈较高的负相关性;LCSM观察发现,在一定脉冲数范围,胞内Ca^(2+)荧光强度随脉冲数的增加而增加,这说明脉冲磁场作用造成细胞生物膜通透性改变,使胞外大量Ca^(2+)跨膜进入胞内。由此推测,由脉冲磁场造成的细胞生物膜通透性的变化,导致胞内Ca^(2+)浓度的升高,这是脉冲磁场具有杀菌作用的重要原因。

The purpose of this paper is to study Ca^2＋ transmembrane behaviors of L. grayi treated by pulsed magnetic fields. Fluo-4/AM fluorescence probe was used to determine the intracellular Ca^2＋ concentration in L. grayi., Mean- while, the measuring method of intracellular Ca2. fluorescence intensities was established, and the changes of intracellular Ca2. concentration was observed by LCSM. Resnhs showed that Fluo-4/AM can load into L. grayi successfully and can be used to determine the intracellular free Ca^2＋ concentration. The optimal loading concentration, time and temperature are were 6 μmol/L, 60 min and 37 ℃, respectively. After being exposed to PMF, intracellular free Ca^2＋ concentration of L. grayi changed and was found to have a negative correlation with the total number of colonies. In addition, LCSM obser- vation showed that Ca^2＋ fluorescence intensities of cytoplasm and nucleus increased, indicating that large amount of exocellular calcium ions got inside the cells. Thus, it can be inferred that PMF might change membrane permeability and increase in intracellular free Ca^2＋ concentration of L. graft, resulting in the cells death finally.