运用蛋白质组学技术研究TNF-α对血管内皮细胞的作用机制

目的:研究TNF-α对培养血管内皮细胞蛋白质表达谱的影响,进一步探讨TNF-α对内皮细胞作用的分子机制,为寻找治疗心血管疾病的潜在靶标奠定基础。方法: 用一氧化氮检测试剂盒检测人脐静脉内皮细胞(HUVECs)释放的一氧化氮的量;用二向电泳、图像分析、质谱鉴定等蛋白质组学技术测定TNF-α作用于血管内皮细胞后引起表达量有显著改变的蛋白质。结果: TNF-α可减少HUVECs释放一氧化氮的量。TNF-α作用HUVECs 6 h后有21个蛋白质表达量显著改变,其中11个蛋白质下调和9个蛋白质上调,并且有一个蛋白蛋只出现在TNF-α刺激的HUVECs中。结论: TNF-α抑制内皮细胞释放一氧化氮可能与其降低eNOS蛋白质表达和增加网钙结合素蛋白质表达有关;TNF-α增加MEKK-3的蛋白表达可能与其增加粘附分子的表达有关;运用蛋白质组学技术发现了内皮细胞中多个新的TNF-α的影响蛋白质,为寻找治疗心血管疾病的潜在靶标奠定了基础。

Proteomic analysis of the effects of tumor necrosis factor- α on endothelial cells

AIM: To investigate the affected proteins by tumor necrosis factor (TNF)-α in endothelial cells, and further explore the potential molecular mechanism of TNF-α on endothelial cells. METHODS: Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by a NO assay kit. Proteomic alterations were analyzed using two-dimensional electrophoresis, and peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: NO production in HUVECs decreased significantly after TNF-α treatement. Proteomics analysis showed 21 protein spots were changed including 9 spots that were increased and 11 spots that were decreased after TNF-α stimulation, and 1 spot was only detected in TNF-α activated cell gels. CONCLUSIONS: Decreased the expression of ecNOS by TNF-α might result in decreased NO production. Up-regulated MAP/ERK kinase 3 expression might imply that TNF-α activates the expression of adhesion molecules. Cytoskeletal protein actin is also involved in TNF-α injuried HUVECs. Proteomic analysis can find some clues for identifying new potential target of TNF-α.