Institute of Cardiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, 310003 Hangzhou, P.R. China.
Int J Mol Med. 2010 Mar;25(3):385-92. doi: 10.3892/ijmm_00000356.
The aim of this study was to determine whether proteasome inhibitor MG132 treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. EPCs were identified as adherent cells double positive for DiLDL-up-take and lectin binding by direct fluorescent demonstrated under a laser scanning confocal microscope. After 7 days in culture, EPCs were stimulated with proteasome inhibitor MG132 in series of final concentrations of 20, 50, 100, 200 nmol/l for 12, 24, 48 h. Cell proliferation and apoptosis were determined, respectively, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide binding assay. Colony-forming capacity was performed by colony assay. EPCs adhesion and migration were assayed with adhesion assay and transwell migration assay, respectively. The expression of endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis, while nitric oxide (NO) generation was detected using the Griess method. It was found that proteasome inhibitor MG132 decreased the number of EPCs and EPC colonies, increased EPC apoptosis, decreased EPC proliferative, adhesive, migration capacity and eNOS/NO production in a concentration- and time-dependent manner. These data indicate that proteasome inhibitor MG132 suppresses the number and function of EPCs, and these actions may involve decreased eNOS/NO production in the EPCs.
本研究旨在探讨蛋白酶体抑制剂 MG132 处理对内皮祖细胞（EPCs）的影响。采用 Ficoll 密度梯度离心法从外周血中分离出单个核细胞（MNC）。通过激光扫描共聚焦显微镜下直接荧光染色，鉴定 EPCs 为 DiLDL 摄取和凝集素结合的双阳性贴壁细胞。培养 7 天后，用一系列终浓度为 20、50、100、200nmol/L 的蛋白酶体抑制剂 MG132 刺激 EPCs12、24、48 小时。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和 Annexin V/碘化丙啶结合法分别测定细胞增殖和凋亡。通过集落形成试验测定集落形成能力。通过黏附试验和 Transwell 迁移试验分别测定 EPCs 黏附和迁移能力。通过 Western blot 分析测定内皮型一氧化氮合酶（eNOS）的表达，通过 Griess 法检测一氧化氮（NO）的产生。结果发现，蛋白酶体抑制剂 MG132 呈浓度和时间依赖性地减少 EPC 数量和 EPC 集落，增加 EPC 凋亡，降低 EPC 增殖、黏附、迁移能力和 eNOS/NO 的产生。这些数据表明，蛋白酶体抑制剂 MG132 抑制 EPCs 的数量和功能，这些作用可能涉及 EPCs 中 eNOS/NO 的产生减少。
