表达绿色荧光蛋白的布鲁氏菌16M和M5侵染小鼠巨噬细胞过程的差异分析

Variance Analysis of Macrophages Infected in the Process with Green Fluorescent Protein-expressing Brucella 16M and M5

通过布鲁氏菌（Brucella）16M（强毒株）和M5（弱毒株）进入小鼠巨噬细胞后产生的荧光强度,结合布鲁氏菌胞内生存能力分析二者侵染小鼠巨噬细胞过程的差异,为布鲁氏菌在细胞内的生存繁殖及其分子机制研究提供理论依据。将绿色荧光蛋白（green fluorescent protein,GFP）布鲁氏菌16M（强毒株）和M5（弱毒株）分不同时间段侵染小鼠巨噬细胞（感染复数为100∶1）,通过胞内生存实验、激光共聚焦显微镜和流式细胞仪观察和检测布鲁氏菌侵入RAW 264.7过程的差异。结果表明,布鲁氏菌16M和M5和GFP的16M和M5侵染RAW264.7的能力不受影响,通过流式细胞仪可以鉴别GFP布鲁氏菌,但是不能确定细菌的存活状态。结合布鲁氏菌16M和M5胞内生存能力分析发现GFP＋细胞的增加与活菌培养计数法（colony forming unit,CFU）数量的减少存在一一对应的关系,并且在侵染初期（5~25 min）16M与M5的荧光强度并没有显著差异。结果提示,布鲁氏菌16M和M5在侵染进入宿主细胞的初期即侵袭能力并没有明显差异,认为造成毒力差异的主要原因是两者的繁殖能力。

To explore Brucella 16M and M5 into the macrophages in mice after fluorescence intensity produced by the combination of Brucella intracellular survival ability to analyze and understand differences in both attack and the ability to reproduce. GFP-labeled 16M （virulent strain） and M5 （weak strain） were divided into different time infect mice macrophage RAW 264.7 （bacteria and cell ratio of 100：1）, use of intracellular survival experiment, laser confocal microscope and flow cytometry for observation and detection. The results showed that Brucella melitensis 16M and M5 and GFP-labeled Brucella melitensis 16M and M5 are not affected into macrophages. By flow cytometry instrument can identify GFP Brucella but can not determine the survival of the bacteria. Combined with Brucella 16M and the M5 intracellular survival ability analyzed, it was found that GFP＋ cells increased with the reduction in the number of CFU exists corresponding relationship, and at the beginning of the Brucella 16M and M5 there were no significant differences with fluorescence intensity. The results suggested that the Brucella 16M and M5 arrive the host cells ability is the same, but have the difference between the vlirulent strain and the weak strain, mainly with the ability of survival and intracellular replication.