肠球菌溶血及非溶血菌株对小鼠巨噬细胞表达TNF-α的影响

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

【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-α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200μg/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-α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-αmRNA表达的差异。【结果】未感染的RAW264.7细胞培养液中检测不到TNF-α。肠球菌溶血株感染组细胞培养上清液中各观测点的TNF-α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-αmRNA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-α炎症因子。

[Objective] To study the influence of hemolytic and non-hemolytic enterococci on TNF-α 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 lh, followed by washing and re-incubation for 24h in complete medium containing 200 μg/mL Ampicillin. TNF-α 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-α mRNA expression by macrophages infected with hemolytic or non-hemolytic enerococci respectively for 6h was compared by RT-PCR. [Results] TNF-α was not detected in culture supernatants from uninfected RAW264.7 cells. The concentrations （pg/mL） of TNF-α 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-α mRNA expression measured by RT-PCR brought about similar results： more TNF-α 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-α.