脂质过氧化诱导培养的内皮细胞表达单核细胞趋化蛋白-1

Lipid Peroxidation Induces Expression of Monocyte Chemoattractant Protein-1 in Cultured Endothelial Cells

在人脐静脉和牛主动脉内皮细胞培养基中加入联胺，引发其脂质过氧化损伤，观察能否诱导内皮细胞表述单核细胞起化蛋白河。用斑点杂交法检测内皮细胞暴露于联胺后其单核细胞趋化蛋白-1mRNA的表达，杂交用的探针为了r32P5’末端标记的寡核苷酸探针。同时，用酶联免疫反应检测内皮细胞暴露于联胺后．其条件培养基中的单核细胞趋化蛋白-1蛋白含量。斑点杂交显示．培养的内皮细胞可表达单核细胞趋化蛋白-1mRNA，暴露于联胺后．其单核细胞趋化蛋白-1mRNA表达水平明显升高。而且，单核细胞趋化蛋白-1mRNA表达水平与联胺的作用时间和浓度均呈正相关。酶联免疫反应显示，各组条件培养基中的单核细胞趋化蛋白-1蛋白含量亦与联胺作用的时间和浓度呈正相关。提示内皮细胞的脂质过氧化损伤可诱导其产生单核细胞起化蛋白-1增加，在动脉粥样硬化发生过程中单核细胞的聚集可能起重要作用。

Aim To observe whether lipid peroxidation injury to cultured human umbilical vein endothelial cells and bovine aortic endothelial cells has effects on the expression of monocyte chemoattractant protein-l (MCP-1) in them.Methods The lipid peroxidation injury to endothelial cells (EC) was induced by exposure to diamide either at a same concentration but for different incubation time or at different concentrations but for a same incubation time. The EC in different groups were collected and their total RNA was extracted by the single-step method. The MCP-1 mRNA expression in EC was examined by dot blotting using a Y32 P-end la beled 35 mer-oligonucleotide probe, Meanwhile, MCP-1 protein content in the media conditioned by the cultured EC exposed to diamide was determined by sandwich ELISA.Results Cultured EC could express MCP-1 mRNA and protein. Dot blotting showed that exposure of EC to diamide at a concentration of 5 μmol/L for 2, 4 and 8 h, respectively, resulted in a 2. 26 fold, a 2. 41 fold and a 2. 72 fold increase in the levels of MCP-1 mRNA expression in EC respectively. as compared to the control group. On the other hand .exposure of EC to diamide at different concentrations (1 μmol/L, 5 μmol/L and 10 μmol/L) for 3 h, resuited in a 2. 22 fold, 2. 97 fold and 3. 32 fold increase in the levels of MCP-l mRNA expression in EC respectiveiy. The results of ELISA were similar to that of dot blotting.Exposure of EC to diamide at a same concentration but for different incubation time resuited in a 2. 18 fold, a 3. 87 fold and a 5. 87 fold increase in MCP-1 protein content in the conditioned media. By contrast, exposure of EC to diamide at different concentration but for a same incubation time resuiteh in a 2.34 fold, a 3. 44 fold and a 4. 6 fold increase in MCP-1 protein content in the conditioned media. Together these results showed that the MCP-1 expression in EC induced by exposure to diamide was dependent on both dose and the incubation time.Conclusion Lipid peroxidation to EC can induce increased production of MCP-1 in the cells and may play an important role in the recruitment of monocytes into the intima in atherogenesis.