LPS-TLR4复合物内化障碍与LPS介导巨噬细胞活化的实验研究

Experimental study of internalization dysfunction of LPS-TLR4 complex on LPS-induced activation of macrophages

目的建立内化障碍的小鼠RAW264.7细胞模型,观察脂多糖(LPS)-Toll样受体4(TLR4)复合物内化障碍对巨噬细胞活化作用的影响。方法应用内化抑制剂单丹磺酰尸胺(MDC),构建内化障碍的小鼠RAW264.7细胞模型;四甲基偶氮唑盐(MTT)检测MDC对巨噬细胞的毒性;应用LPS刺激内化障碍巨噬细胞,流式细胞仪、激光共聚焦显微镜检测LPS-TLR4复合物内化抑制情况;酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)蛋白的表达;实时PCR法检测TNF-α及IL-6 mRNA的表达。结果应用化学抑制剂MDC,在流式细胞仪和激光共聚焦显微镜下,均观察到RAW264.7细胞中LPS-TLR4复合物的内化被显著抑制,成功建立了内化障碍的模型RAW264.7细胞;反映该细胞活化的指标IL-6在蛋白水平和核酸水平均受到明显抑制(P<0.05),而TNF-α的表达在蛋白水平和核酸水平均无显著抑制。结论 LPS-TLR4复合物内化与LPS介导巨噬细胞活化密切相关,内化障碍条件下,由LPS介导的细胞活化表现为对IL-6的释放受到显著抑制,而对TNF-α的释放则抑制不显著。

Objective Treatment with inhibitor of internalization to establish the cell model of internalization dysfunction and examine the effect on internalization of LPS-TLR4 complex in RAW264.7 cell model.Methods Utilizing chemical inhibitor,Monodansylcadaverine,to establish RAW264.7 cells model of internalization dysfunction;MTT assay MDC on macrophage toxicity;The inhibition effectiveness on LPS-TLR4 complex internalization was examined by flow cytometry and confocal laser scanning microscopy;ELISA to detect TNF-α,IL-6 protein expression;real-time PCR to detect TNF-α mRNA,IL-6 mRNA expression.Results MDC prevented LPS-induced internalization of LPS and TLR4 complex and thus allowed the establishment of RAW264.7 cells model of internalization dysfunction;real-time PCR and ELISA data indicated that pretreatment of cells with100 μmol/L MDC dramatically inhibited the expression of IL-6（P0.05）;TNF-α protein and nucleic acid levels were not significant inhibited.Conclusion Internalization of LPS-TLR4 complex is closely related to macrophage activation.Under the condition of internalization hindrance,LPS-induced cell activation displays significant inhibition of IL-6 release,but no such effects on TNF-α release.