内毒素脂多糖诱导培养的人脐静脉内皮细胞表达巨噬细胞炎性蛋白-1α

Lipopolysaccharide induces expression of macrophage inflammatory protein-1α in human umbilical vein endothelial cells

目的 了解内毒素脂多糖是否诱导人脐静脉内皮细胞 (HUVEC)表达巨噬细胞炎性蛋白 1α(MIP 1α)。方法 使培养的HUVEC暴露于不同浓度的脂多糖 ,用地高辛标记的MIP 1αcDNA探针与HUVEC的总RNA进行斑点杂交 ,并与HUVEC进行原位杂交 ,同时用HUVEC的总RNA与MIP 1α引物的混合物进行逆转录 聚合酶链反应 (RT PCR) ,以检测HUVEC的MIP 1αmRNA的表达 ;再者 ,将培养的HUVEC用MIP 1α单克隆抗体进行细胞酶联免疫吸附试验 (ELISA) ,以检测其MIP 1α蛋白表达。结果 斑点杂交显示 ,暴露于浓度为 1μg/ml和 10 μg/ml脂多糖时HUVECmRNA在硝酸纤维素膜上斑点的积分吸光度 (A)值分别为 1 490和 3 3 10 ,分别为对照组 (0 775)的1 97倍和 4 3 8倍。原位杂交显示 ,当HUVEC暴露于浓度为 1μg/ml脂多糖后 ,其MIP 1αmRNA表达与对照组相比有明显增加 ,方差分析表明 ,差异有非常显著性 (F =14 2 83 ,P <0 0 1)。但当其暴露于 10 μg/ml脂多糖后 ,其MIP 1αmRNA表达则较低。RT PCR显示 ,暴露于浓度为 1、5和 10 μg/ml脂多糖时HUVEC的MIP 1αmRNA表达分别为对照组的 1 65倍、2 86倍和 1 2 6倍。细胞ELISA显示 ,各组HUVEC暴露于脂多糖后 ,其MIP 1α蛋白表达均明显增加 ,尤以 5μg/ml脂多糖组最为显著。方差分析表明 ,差?

Objective To understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1α (MIP-1α) mRNA and protein in human umbilical vein endothelial cells (HUVECs). Methods The expression of MIP-1α mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1α cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1α mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1α protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1α antibody. Results Dot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 μg/ml and 10 μg/ml which we re 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 μg/ml led to a significant increase in the MIP-1α mRNA expression in HUVECs as compared to the control group (F=142.83, P<0.01),whereas the MIP-1α mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 μg/ml. RT-PCR revealed that the expression of MIP-1α mRNA in HUVECs were 1.65-, 2.86- and 1.26- fold over that of the control group when exposed to LPS at the concentrations of 1 μg/ml, 5 μg/ml and 10 μg/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1α protein was strongly increased, especially in the 5 μg/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F=15.36, P<0.05). Conclusions LPS may induce a high level of MIP-1α mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.