人肠上皮细胞耐受内毒素的分子机制

The molecular mechanism by which human intestinal epithelial cells tolerate lipopolysaccharide

目的 观察人肠上皮细胞对内毒素刺激的反应性 ,探讨其耐受内毒素的分子机制。方法 分别用凝胶迁移阻滞法 (EMSA)和酶联免疫吸附分析 (ELISA)法检测系列浓度的内毒素刺激人小肠上皮细胞株 (HIC) 1h后细胞核转录因子 (NF κB)的活性变化和刺激 18h后细胞白细胞介素 8(IL 8)的分泌水平。用核糖核酸酶保护法 (RPA )检测HIC内毒素相关受体———Toll样受体 4(TLR4)、CD14和MD 2mRNA的表达。将转入TLR4、CD14和MD 2表达质粒的HIC经内毒素刺激后 ,检测细胞NF κB和IL 8水平的变化。结果 用系列浓度的内毒素刺激HIC后 ,均检测不到NF κB的活性和IL 8的分泌 ;HIC不表达TLR4、CD14和MD 2mRNA ;转染TLR4、CD14和MD 2质粒的HIC受脂多糖 (LPS)刺激后 ,可检测到较强的NF κB活性和明显的IL 8分泌(2 17.3 3± 3 3 .2 1)ng/L。 结论 HIC呈内毒素无反应性 ,其表面内毒素相关受体TLR4、CD14和MD 2不表达是其耐受内毒素作用的重要分子机制。

Objective To observe the responsiveness of human intestinal epithelial cells (HIC) to lipopolysaccharide (LPS) stimulation,and to explore the molecular mechanism by which HICs tolerate LPS.Methods The activation of NF-κB in HICs which were stimulated by a series of concentrations of LPS at 1 h was detected by EMSA and IL-8 in supernatants of HICs was measured by ELISA at 18 h following LPS stimulation.The expression of TLR4,CD14 and MD-2 mRNA in HIC was detected by RNase protection assay.After the HIC transfected with TLR4,CD14 and MD-2 plasmids were stimulated by LPS at 1 h and 18 h,NF-κB and IL-8 were analyzed respectively.Results The activation of NF-κB and the secretion of IL-8 were not detectable after HICs were treated with LPS,and HICs did not express TLR4,CD14 and MD-2 mRNA.However,the activation of NF-κB and the secretion of IL-8 were detectable in HICs when simultaneously transfected with TLR4,CD14 and MD-2 plasmids in response to LPS.Conclusion There was no response of HICs to LPS stimulation,and no expression of TLR4,CD14 and MD-2 on HICs may be an important molecular mechanism by which they tolerated to lipopolysaccharide.