L-精氨酸和尼莫地平对脂多糖诱导的内皮细胞表达单核细胞趋化蛋白-1的影响

The effects of L-arginine and nimodipine on the expression of monocyte chemoattractant protein-1 in endothelial cells induced by lipopolysaccharide

目的 观察L 精氨酸和尼莫地平对脂多糖 (LPS)诱导的内皮细胞 (EC)的单核细胞趋化蛋白 1(MCP 1)mRNA和蛋白表达的影响。方法 在EC培养基中分别加入LPS、LPS +L 精氨酸和LPS+尼莫地平 ,培养 8小时 ,用异硫氢酸胍法提取细胞的总RNA ,用γ 32 P末端标记的MCP 1寡核苷酸探针进行Dotblot分析 ,检测EC的MCP 1mRNA的表达。同时用夹心酶联免疫吸附实验检测各组EC条件培养基 (CM)中的MCP 1蛋白含量。用免疫细胞化学方法检测EC的MCP 1蛋白的表达。结果 斑点杂交显示 ,EC暴露于LPS后 ,其斑点的积分吸光度 (A)值为 3 14 ,对照组 (条件培养基 )为 1 2 4 ,而LPS +L 精氨酸组和LPS +尼莫地平组的积分A值分别为 1 4 3和 1 65。ELISA检测结果显示 ,LPS组的CM中MCP 1蛋白含量为 ( 6 2 9± 0 5 3 )ng/ml,对照组为 ( 2 3 2± 0 .16)ng/ml,两者比较F =3 0 9.81,P<0 .0 1,而LPS +L 精氨酸组和LPS +尼莫地平组分别为 ( 7 63± 0 .3 1)及 ( 2 75± 0 .3 7)mg/ml,与LPS组比较F =3 0 9.81,P <0 .0 1。免疫细胞化学显示 ,MCP 1为胞浆表达 ,LPS组EC对MCP 1多克隆抗体呈强阳性反应 ,而对照组、L 精氨酸组和尼莫地平组EC胞浆中的MCP 1表达呈弱阳性。结论 L 精氨酸和尼莫地平能明显抑制LPS诱导的ECMCP 1的表达。

Objective To examine the effects of L arginine and nimodipine on the expression of monocyte chemoattractant protein 1 (MCP 1) mRNA and protein induced by lipopolysaccharide (LPS) in endothelial cells (ECs). Methods After 8 hour exposure to LPS, LPS+L arginine and LPS+nimodipine respectively, the total RNA in ECs was extracted by the guanidinium isothiocyanate method. The MCP 1 mRNA expression in ECs was examined by Dot blot analysis using a γ 32 P end labeled 35 mer oligonucleotide probe of MCP 1. MCP 1 protein in the EC conditioned media (EC CM) was determined by sandwich ELISA for each group. The expression of MCP 1 protein in ECs was examined immunocytochemically. Results Dot blotting showed that after exposure to LPS, the integral absorbance ( A ) value of the dots of MCP 1 mRNA on the nitrocellulose membrane were 3.14, which was 2.5 fold as much as that in the control group (A=1.24). The A values of the LPS+L arginine group and the LPS+nimodipine group were 1.43 and 1.65, respectively. ELISA showed that the MCP 1 protein content in the EC CM of the LPS group was 6.29±0.53 ng/ml, which was markedly higher than that of the control group (2.32±0.16) ng/ml ( P <0.01). The MCP 1 protein content in the EC CM of the LPS+L arginine group and the LPS+nimodipine group was significantly lower than that of the LPS group ( P <0.01). Furthermore, the ECs in LPS group showed a strong immunoreactivity for the polyclonal MCP 1 antibody, whereas the ECs in control, L arginine and nimodipine groups revealed a weak postive immunostaining. Conclusion L arginine and nimodipine can markedly inhibit the expression of MCP 1 in ECs induced by LPS.