晚期糖基化终产物刺激内皮细胞分泌单核细胞趋化蛋白-1信号传导途径  

Advanced glycation end products stimulate human endothelial cells to produce monocyte chemoattractant protein-1

作　　者：郭志坚[1] 侯凡凡[1] 梁敏[1] 王力[1] 张训[1] 刘志强[1] 

机构地区：[1]第一军医大学南方医院肾内科

出　　处：《中华医学杂志》2003年第12期1075-1079,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目 ( 3 9970 3 41);广东省自然科学基金重点资助项目 ( 0 13 0 76);广东省团队项目基金资助项目 ( 10 717)

摘　　要：目的　研究晚期糖基化终产物 (AGE)修饰蛋白对人内皮细胞分泌单核细胞趋化蛋白 1(MCP 1)的影响及其作用的信号传导途径。方法　将培养的人脐静脉内皮细胞 (HUVEC)和人内皮细胞株ECV30 4与不同浓度AGE修饰的人血清白蛋白 (AGE HSA)或牛血清白蛋白 (AGE BSA)共同培养。用Western印迹及酶联免疫吸附法 (ELISA)检测MCP 1蛋白合成及分泌 ,用逆转录PCR(RT PCR)法检测MCP 1mRNA表达 ,用流式细胞术观察细胞内氧化应激 ,用免疫沉淀 激酶活性测定法分析细胞p38丝裂素活化蛋白激酶 (p38 MAPK)活性。结果　AGE HSA和AGE BSA以时间和剂量依赖的方式上调内皮细胞MCP 1mRNA和蛋白的表达并诱导细胞氧化应激和活化p38 MAPK。 5 0 μg/mlAGE HSA与HUVEC共同培养 12h ,使细胞上清中的MCP 1浓度由 4 8 3pg/μg± 0 6pg/μg蛋白上升至 14 8 1pg/μg± 12 6pg/μg蛋白 (P <0 0 1)。5 0 μg/mlAGE HSA与HUVEC共同孵育 30min ,使p38 MAPK的磷酸化活性升高 91%± 14 % (P <0 0 1)。抗氧化剂或p38通路特异阻断剂SB 2 0 35 80能够阻断AGE修饰蛋白诱导的MCP 1表达。结论　AGE修饰蛋白能够通过p38 MAPK信号传导途径上调内皮细胞分泌MCP 1,这一作用是经氧化应激机制介导。Objective To investigate the effects of advanced glycation end products (AGE) on secretion of monocyte chemoattractant protein-1(MCP-1) by human endothelial cells and its signal transduction pathway. Methods Human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were cultured in vitro with indicated concentration of AGE modified human serum albumin (AGE-HSA) or AGE modified bovine serum albumin (AGE-BSA). The production of MCP-1 was evaluated by Western blotting and enzyme-linked immunoadsorbent assay (ELISA). The MCP-1 mRNA expression was assayed by reverse-transcription polymerase chain reaction (RT-PCR). Intracellular oxidative stress was detected by flow cytometry. The phosphorylation activity of cellular p38 mitogen-activated protein kinase (p38-MAPK) was analyzed by Western blotting using a phospho-specific antibody. Results AGE-HSA and AGE-BSA, but not their unmodified form, upregulated the expression of MCP-1 mRNA and protein dose- and time-dependently. The MCP-1 concentration in the supernatant of HUVECs incubated with 50 μg/ml AGE-HSA for 12 hours increased from 48.3 pg/μg±0.6 pg/μg protein to 148.1 pg/μg±12.6 pg/μg protein (P<0.01). AGE modified proteins were associated with enhanced oxidative stress and p38-MAPK phosphorylation activity. Incubation of HUVECs with 50 μg/ml AGE-HSA for 30 minutes resulted in increase of p38-MAPK phosphorylation activity by 91%±14% (P<0.01). Antioxidant or SB 203580, a specific inhibitor of p38, could block the over-expression of MCP-1. Conclusion AGE modified proteins stimulate endothelial cells to produce MCP-1 through activation of the p38 signal pathway. This effect may contribute to the pathogenesis of atherosclerosis seen in AGE-associated diseases.

关 键 词：糖基化终产物 内皮细胞 单核细胞趋化蛋白-1 信号传导途径 氧化应激 细胞培养 动脉粥样硬化 

分 类 号：R543.5[医药卫生—心血管疾病]