糖基化终产物对大鼠主动脉平滑肌细胞巨噬细胞炎性蛋白-1α表达的影响

Effect of advanced glycosylation end products on protein and mRNA expression of macrophage inflammatory protein-1α in cultured vascular smooth muscle cells

目的 探讨糖基化终产物 (AGEs)对大鼠主动脉平滑肌细胞巨噬细胞炎性蛋白 1α(MIP 1α)mRNA及蛋白表达的影响。 方法 将培养的大鼠主动脉平滑肌细胞用不同浓度 (10 0、2 0 0、4 0 0mg/L)的AGEs孵育 2 4h及同一浓度 (4 0 0mg/L)AGEs孵育 0、12、2 4、36h ,采用RT PCR方法及流式细胞仪检测MIP 1αmRNA及蛋白的表达水平。 结果 对照组平滑肌细胞内MIP 1α呈弱表达 ,10 0、2 0 0、4 0 0mg/LAGEs培养平滑肌细胞 2 4h显著增高MIP 1αmRNA的表达 ,其电泳条带相对积分吸光度值分别为对照组的 1 36、1 75、2 4 5倍 (P <0 0 5 ) ;10 0、2 0 0、4 0 0mg/LAGEs孵育2 4h后 ,各组平滑肌细胞MIP 1α蛋白的平均荧光强度分别为 197± 3、2 6 0± 6及 375± 6 ,分别为对照组(15 8± 4 )的 1 2 4、1 6 3、2 37倍 (P <0 0 5 )。 4 0 0mg/LAGEs培养 12、2 4、36h后 ,各组平滑肌细胞MIP 1αmRNA的表达也明显增高 ,其电泳条带相对积分吸光度值分别为 0h组的 1 5 2、2 38、2 5 3倍(P <0 0 5 ) ;4 0 0mg/LAGEs孵育 12、2 4、36h后 ,各组平滑肌细胞MIP 1α蛋白的平均荧光强度分别为 2 4 4± 5、375± 6及 4 2 5± 3,分别为 0h组 (170± 5 )的 1 4 3、2 2 1、2 4 9倍 (P <0 0 5 )。

Objective To investigate the effect of advanced glycosylation end products (AGEs) on protein and mRNA expression of macrophage inflammatory protein -1α (MIP-1α) in cultured vascular smooth muscle cells(VSMCs). Methods VSMCs were cultured with AGEs at different concentrations(100, 200, 400mg/L) for 24 h and at a concentration of 400 mg/L for 0, 12, 24 and 36 h.The levels of mRNA and protein expression of MIP-1α in cultured VSMCs were detected by RT-PCR and flow cytometry. Results RT-PCR showed that after exposure of VSMCs to AGEs at different concentrations(100, 200, 400 mg/L) for 24 h, the expression of MIP-1αmRNA was increased 1.36-fold, 1.75-fold and 2.45-fold respectively compared with BSA group(P<0.05); meanwhile, after exposure of VSMCs to AGEs at a concentration of 400 mg/L for 12, 24 and 36 h, the expression of MIP-1αmRNA in VSMCs was increased 1.52-fold, 2.38-fold and 2.53-fold respectively compared with 0 h group(P<0.05). Flow cytometry showed that after exposure of VSMCs to AGEs at different concentrations(100, 200, 400 mg/L) for 24 h, resulted in a 1.24-fold, a 1.63-fold and a 2.37-fold increase in the expression of MIP-1α protein in VSMCs compared with BSA group(P<0.05), and the average fluorescent intensity of MIP-1α mRNA expression in VSMCs were 197±39, 260±6 and 375±6(BSA group,158±4). Exposure of VSMCs to AGEs at a concentration of 400 mg/L for 12, 24 and 36 h, MIP-1α in VSMCs was increased 1.43-fold, 2.21-fold and 2.49-fold compared with 0 h group(P<0.05), and the average fluorescent intensity of MIP-1αmRNA expression in VSMCs were 244±5, 375±6 and 425±35(0 h group,170±5). Conclusions The results suggest that AGEs induce a high expression of MIP-1αmRNA and protein in cultured VSMCs in a dose-dependent and time-dependent manner.