肠球菌溶血及非溶血菌株对小鼠巨噬细胞表达TNF-a的影响

Influence of hemolytic and non-hemolytic enterococci on TNF-aexpression in murine macrophage cells

摘要:

【目的】探讨肠球菌溶血菌株及非溶血菌株对小鼠巨噬细胞RAW264.7表达TNF-а的影响。【方法】用多粘菌素B抑制排除内毒素污染对实验的影响。肠球菌溶血菌株、非溶血菌株各11株，以菌/细胞比 30∶1 感染RAW264.7细胞1 h，加入200 mg/mL氨苄青霉素继续培养24 h，分别于感染后3、6、9、24 h，用ELISA方法检测不同观测点细胞培养液中肿瘤坏死因子TNF-a的含量，并用逆转录-聚合酶链反应方法（RT-PCR）比较肠球菌溶血、非溶血菌株感染6 h后TNF-a mRNA表达的差异。【结果】未感染的RAW264.7细胞培养液中检测不到TNF-a。肠球菌溶血株感染组细胞培养上清液中各观测点的TNF-a的平均含量 (pg/mL) 均比非溶血株性组高。经t检验，P<0.01，差别有显著性。RT-PCR法检测其mRNA的表达也有相同结果：TNF-a mRNA在肠球菌溶血株感染细胞中的相对表达量比非溶血株感染的细胞高，经t检验，P<0.05，差别有统计学意义。【结论】肠球菌溶血株比非溶血株更能促进小鼠巨噬细胞RAW264.7产生TNF-а炎症因子。

Abstract:

[Objective] To study the influence of hemolytic and non-hemolytic enterococci on TNF-a expression in Murine macrophage cells. [Methods] The possibility of endotoxin contamination in the cell culture was excluded by polymyxin-B inhibition. RAW264.7 were infected with hemolyticor non-hemolytic enerococci at bacteria: cell ratio of 30:1 for 1h, followed by washing and re-incubation for 24h in complete medium containing 200 mg/mL Ampicillin. TNF-a concentrations in culture supernatants at different time intervals after infection for 3 h, 6 h, 9 h and 24 h were measured by ELISA. TNF-a mRNA expression by macrophages infected with hemolytic or non-hemolytic enerococci respectively for 6h was compared by RT-PCR. [Results] TNF-a was not detected in culture supernatants from uninfected RAW264.7 cells. The concentrations (pg/mL) of TNF-a present in culture supernatants from RAW264.7 cells stimulated by hemolytic enterococci were significantly higher than that stimulated by non-hemolytic enterococci at all time intervals tested, p<0.01, Student t-test. TNF-a mRNA expression measured by RT-PCR brought about similar results: more TNF-a mRNA was expressed in RAW 264.7 cells stimulated with hemolytic enterococci as compared with non-hemolytic enterococci stimulation (p<0.05, Student t-test). [Conclusion] hemolytic enterococci promoted the generation of inflammatory factor TNF-a.