枸橼酸对内毒素刺激下单核细胞细胞因子分泌的影响

Effect of citrate on lipopolysaccharide-induced monocyte cytokines release

目的:通过体外细胞培养,观察不同浓度枸橼酸对内毒素(LPS)诱导的单核细胞分泌细胞因子功能的影响。方法:采用标准方法体外培养人类单核细胞株(THP-1),加入适量枸橼酸三钠(TSC)使其体外培养液TSC浓度分别为0、0.5 mmol/L、1.0 mmol/L,并给予不同浓度LPS(0.05μg/ml、0.1μg/ml、1μg/ml)刺激,分别于刺激后6h、12h、24h、48h用CCK-8试剂盒测定细胞活性、ELISA试剂盒测定上清液细胞因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平及血气分析仪测定培养基内离子钙(Ca^(2+))水平。使用正常培养条件下的单核细胞做为对照组。结果:不同浓度TSC对单核细胞活性无抑制作用(P>0.05),但对不同时间点LPS诱导的单核细胞分泌细胞因子(TNF-α和IL-6)功能有一定影响,培养基TSC浓度0.5 mmol/L和1.0 mmol/L时,培养12h和24h TNF-α的分泌量显著高于对照组(P<0.01);TSC浓度为0.5 mmol/L时,IL-6在24h开始出现分泌增高趋势,48h达到峰值(P<0.05),TSC浓度1.0 mmol/L显著抑制IL-6分泌(P<0.01)。结论:本实验采用的TSC浓度不存在细胞毒性,单核细胞在此浓度下,受LPS刺激时表现出TNF-α分泌亢进的现象,而IL-6的分泌则表现出TSC低浓度下亢进,高浓度下受抑制的趋势。

Objective： To investigate the effects of various concentrations of citrate on cytokines release by monocyte under stimulation of lipopolysaccharide（ LPS） in vitro. Methodology： Human acute monocyte leukemia cell line（ THP-1） cells were cultured in vitro with different concentrations of trisodium citrate（ TSC,0,0. 5,1. 0 mmol/L）,and were stimulated with different concentrations of LPS（ 0,0. 05,0. 1,1 μg/ml）.Cell samples and supernatants were collected 6 h,12 h,24 h and 48 h after cell culture. The monocyte viability,concentrations of interleukine-6（ IL-6）,tumor necrosis factor-α（ TNF-α） and ionized calcium（ Ca^（2＋）） were observed. Results： Various concentrations of citrate did not inhibit the viability of monocyte（ P0. 05）.The monocyte TNF-α production after LPS stimulation was augmented with 0. 5 mmol/L and 1 mmol/l citrate at 12 h and 24 h when compared with no citrate（ P0. 01）.The production of IL-6 was augmented with0. 5 mmol/L citrate at 24 h and 48 h（ p〈0. 05）,but was inhibited with 1. 0 mmol/L citrate when compared with no citrate（ P0. 01）. Conclusion： Citrate in the investigated concentrations has no cytotoxicity to monocyte.After stimulation by LPS,production of TNF-α was augmented with citrate,while production of IL-6 was augmented only with low concentration of citrate but was inhibited with high concentration of citrate.