过氧化氢碳酰胺诱导血管内皮细胞衰老模型的建立和验证

The establishment and validation of an endothelial cell senescence model induced by carbamide peroxide

目的用过氧化氢碳酰胺（carbamide peroxide，CP）作为诱导剂，诱导猪髂动脉内皮细胞（pig iliac endothelium cells，PIEC）衰老，建立体外内皮细胞衰老模型。方法MTT法及DAPI染色法确定CP对PIEC作用的最佳诱导浓度。细胞经过诱导后，形态学观察、MTT法、EdU掺人实验、SA-β—gal染色以及细胞迁移实验分析诱导细胞增殖活性、衰老比例以及迁移能力改变。PI单染流式细胞术分析诱导前后细胞周期的分布变化。结果40μmol／L为CP诱导内皮细胞的最佳浓度，诱导1h后，细胞变大变圆，呈典型的衰老表现，增殖活性和迁移能力下降，SA-β—gal染色显示衰老细胞比例明显增大，流式细胞分析显示大量细胞阻滞于G0／G1期。结论CP能够体外诱导血管内皮细胞衰老，是一种安全高效的体外细胞衰老诱导剂。

Objective To establish an in vitro pig iliac artery endothelial cells （PIECs） senescence model using carbamide peroxide （CP）. Methods MTT assay and DAPI staining were used to define the optimal concentration of CP for inducing to the PIECs senescence model. Cellular morphology, MTT assay, EdU labeling, SA-β-gal staining and cell scratch test were performed to analyze the cell growth kinetic, proliferative activity, aging ratio and migratory activity difference post CP induction. PI signal staining flow cytometry was used to analyze the cell cycle distribution difference of cells before and after CP induction. Results The optimal CP concentration was 40 μmol/L to induce PIECs senescence. After 1 h treatment with 40 μmol/L CP, the PIECs presented typical aging form with lager and more rounded shapes. Compared with control group, the proliferative activity and the migratory distance of CP group were significantly decreased; the SA-β-gal staining positive ratio was significantly increased; the data of mitotic cycle distribution with flow cytometry analysis showed that most ceils were arrested at G1/G0 phase. Conclusion CP could efficiently induce pig iliac artery endothelial cell senescence in vitro.