雷帕霉素对内皮祖细胞增殖、黏附及迁移能力影响的实验研究

目的药物洗脱支架可抑制支架处血管内膜再内皮化,但对骨髓源性内皮修复作用的影响尚不清楚,为此观察了雷帕霉素对内皮祖细胞(endothelial progenitor cells,EPC)增殖、黏附及迁移的影响。方法采用密度梯度离心法从大鼠骨髓获取单个核细胞,将其接种在纤维连接素包被培养板,加入不同浓度雷帕霉素(0.01～100ng/ml),培养12天后,激光共聚焦显微镜鉴定FITC-UEA-Ⅰ和DiI-acLDL双染阳性细胞为正在分化的EPC,并在倒置荧光显微镜下计数。收集培养8天贴壁细胞,分别加入不同浓度雷帕霉素(0.1～200ng/ml)培养12～96h。然后分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验观察EPC的增殖、迁移和黏附能力。结果雷帕霉素显著抑制骨髓单个核细胞分化为EPC,0.1ng/ml浓度雷帕霉素作用12天,EPC减少了(59.3±5.8)%(P<0.01)。雷帕霉素也显著抑制EPC增殖,其抑制作用随雷帕霉素浓度增加及作用时间延长而增加,1ng/ml浓度雷帕霉素作用24h使EPC数量减少(41.5±2.2)%(P<0.01)。雷帕霉素也显著抑制EPC的黏附和迁移能力。结论雷帕霉素可抑制骨髓单个核细胞分化为EPC,并抑制EPC的增殖及迁移能力。

Sirolimus inhibits the differentiation, proliferation and migration of endothelial progenitor cells in vitro

OBJECTIVE: To investigate the effect of sirolimus on differentiation, proliferation, adhesion and migration of endothelial progenitor cells (EPC) in vitro. METHODS: (1) Mononuclear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured on fibronectin-coated culture dishes with or without sirolimus (0.01 - 100 ng/ml) for 12 days. (2) After 8 days cultured, attached cells were treated with sirolimus (0.1 - 200 ng/ml) or vehicle for various time points (12 h, 24 h, 48 h and 96 h). EPC were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal immunofluence microscope. EPC proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively. RESULTS: EPC number differentiated from MNC at 12 days was significantly lower in sirolimus treated cells in a dose-dependent manner than that of vehicle-treated cells. Sirolimus also significantly inhibited the proliferative, migratory and adhesive capacity of EPC in a time and dose dependent manner. CONCLUSION: Present results suggested that sirolimus could inhibit EPC differentiation from MNC and reduce the proliferation, migration and adhesion capacities of EPC.