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

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10μg／,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3．310,分别为对照组(0．775)的1．97倍和4．38倍。原位杂交显示,当HUVEC暴露于浓度为1μg／,ml脂多糖后,其MIP-1α mRNA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=142．83,P＜0．01)。但当其暴露于10μg／ml脂多糖后,其MIP-1α mRNA表达则较低。RT-PCR显示,暴露于浓度为1、5和10μg／ml脂多糖时HUVEC的MIP-1α mRNA表达分别为对照组的1．65倍、2．86倍和1．26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,差异有显著性(F=15．36,P＜0．05)。结论内毒素脂多糖可诱导培养的HUVEC表达高水平的MIP-1αmRNA和蛋白,从而在动脉内膜的单核／巨噬细胞的募集起重要作用。

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

OBJECTIVE: To understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs). METHODS: The expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha 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 micro g/ml and 10 micro g/ml which were 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 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha 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 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/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-1alpha 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.