羊种布鲁菌强毒株和弱毒株对巨噬细胞影响的比较

Influences of Brucella melitensis with different virulence on macrophages

目的 研究毒力不同的两种光滑型羊种布鲁菌在巨噬细胞内存活及诱导细胞因子能力的差异，建立体外布鲁菌毒力检测方法。方法 建立羊种布鲁菌强毒株16M和弱毒株M5侵染小鼠巨噬细胞RAW264.7的细胞模型，采用酶联免疫吸附试验（ELISA）法检测布鲁菌侵染后4、8、24 h，RAW264.7细胞上清培养液中白细胞介素（IL）-1β、IL-6和IL-12的表达水平；涂平板计数法检测布鲁菌侵染后2、4、8、24、48 h，RAW264.7细胞内布鲁菌的数量变化。结果 与对照组［（35.375 ± 1.132）、（35.907 ± 1.025）ng／L］比较，布鲁菌16M和M5侵染组在4 h［（41.896 ± 0.575）、（38.561 ± 0.305）ng／L］、8 h［（38.943 ± 0.097）、（38.159 ± 0.236）ng／L）时IL-6的表达量明显增加（P均 〈 0.05）；与对照组［（341.365 ± 14.672）、（382.235 ± 11.151）ng／L］比较，布鲁菌16M和M5侵染组在4 h［（414.865 ± 4.844）、（447.035 ± 12.409）ng／L］和8 h［（435.445 ± 3.684）、（453.594 ± 4.942）ng／L］时IL-12的表达量明显增加（P均 〈 0.05）。且在侵染4 h时，16M侵染组IL-6的表达量明显高于M5侵染组（P 〈 0.05）。各侵染时间IL-1β的表达量组间比较差异无统计学意义（P均 〉 0.05）。布鲁菌16M和M5侵入细胞的数量无明显差别，但16M在细胞内存活及复制增殖的能力明显强于M5。结论 布鲁菌16M在RAW264.7细胞内的存活能力强于M5，通过测量不同布鲁菌在细胞内存活情况来鉴别其毒力强弱的方法具有可行性。

Objective The survival and replication of two Brucella melitensis （B.melitensis） with different virulence in Raw 264.7 cell and their ability of inducing cytokine were compared to provide basis for establishing a method to identify virulence of Brucella. Methods B.melitensis 16M and M5 were used to infect Raw 264.7 cells and the amount of interleukin （IL）-1β, IL-6 and IL-12 in culture supernatants were detected via enzyme-linked immunosorbent assay at 4, 8 and 24 h after infection. We also evaluated the presence of colony-forming units （CFU） at 2, 4, 8, 24 and 48 h after infection via plate count method. Results The secretion of IL-6 in 16M and M5 groups at 4 h [（41.896 ± 0.575）, （38.561 ± 0.305） ng/L] and 8 h [（38.943 ± 0.097）, （38.159 ± 0.236） ng/L] after infection were higher than those of the control [（35.375 ± 1.132）, （35.907 ± 1.025） ng/L, all P 〈 0.05]. The secretion of IL-12 in 16M and M5 groups at 4 h [（414.865 ± 4.844）, （447.035 ± 12.409） ng/L] and 8 h [（435.445 ± 3.684）, （453.594 ± 4.942） ng/L] after infection were higher than those of the control [（341.365 ± 14.672）, （382.235 ± 11.151） ng/L, all P 〈 0.05]. And the difference of the secretion of IL-6 between 16M and M5 groups at 4 h after infection was significant （P 〈 0.05）. There was no significant difference in the secretion of IL-1β between the groups at 4, 8 and 24 h after infection （all P 〉 0.05）. A similar numbers of 16M and M5 were internalized while much more M5 organisms were killed within 8 hours after uptaken. Survived 16M exhibited a higher ability of replication than M5 in Raw 264.7 cells. Conclusion The intracellular viability of 16M is stronger than that of M5 and it indicates that the surviving and replication ability detection of Brucella in macrophages could be a feasible method to identify the virulence of different Brucella strains.