聚肌胞对人脐血内皮祖细胞增殖和凋亡的影响及其机制

Effect of PolyI∶C on the Proliferation and Apoptosis of Human Umbilical Cord Blood-derived Endothelial Progenitor Cells and Its Mechanism

目的观察Toll样受体3(TLR3)的配体聚肌胞(Poly I∶C)对人脐血内皮祖细胞(EPC)增殖、凋亡的影响并阐明其具体机制。方法采用不同浓度的聚肌胞持续干预EPC,通过流式细胞术检测聚肌胞对EPC细胞周期时相分布及细胞凋亡的影响。利用CCK-8细胞增殖试验分别检测肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)对细胞凋亡的影响,以及含半胱氨酸的天冬氨酸蛋白水解酶Caspase 8、Caspase 9的抑制剂、IL-1β受体的中和抗体对聚肌胞诱导EPC凋亡的作用。结果与对照组相比,较高浓度的聚肌胞(1、10 g/L)显著减少S期和G2/M期细胞比例,并通过下调细胞周期蛋白A、B1、D1、E的表达阻滞细胞周期于G0/G1期;同时聚肌胞还呈剂量依赖性诱导EPC凋亡。Caspase 8和Caspase 9的抑制剂并不能抑制聚肌胞诱导的细胞凋亡;TNF-α对EPC的凋亡率无影响;IL-1β呈剂量依赖性诱导EPC凋亡;而使用IL-1β受体的中和抗体anti-IL-1R1预先封闭IL-1β的结合位点后,再加入聚肌胞干预,结果发现高浓度的anti-IL-1R1可以部分抑制聚肌胞诱导的细胞凋亡。结论高浓度的聚肌胞通过将细胞周期阻滞于G0/G1期并诱导细胞凋亡从而抑制EPC增殖。聚肌胞活化TLR3后通过上调IL-1β的表达诱导EPC凋亡,而内、外源性细胞凋亡途径及TNF-α均不参与聚肌胞诱导的细胞凋亡。

Aim To analyze the effect of Toll like receptor 3（TLR3） agonist Poly I ∶ C on the proliferation and apoptosis of human umbilical cord blood-derived endothelial progenitor cells（EPC） and its mechanism.Methods Endothelial progenitor cells were treated with different concentrations of Poly I ∶ C sequentially,and then the phase of cell cycle and cell apoptosis were tested by flow cytometry.CCK-8 assay was used to detect the effect of TNF-α and IL-1β on the apoptosis of endothelial progenitor cells,and the effect of Caspase 8 inhibitior,Caspase 9 inhibitior and IL-1 receptor 1neutralizing antibody（anti-IL-1R1） on Poly I ∶ C-induced apoptosis.Results Compared with the control group,Poly I∶ C at high concentrations of 1 and 10 g/L significantly decreased the proportion of cells in S phase and G2/M phase.Moreover,Poly I ∶ C down-regulated the gene expression of cyclin A,B1,D1 and E,inducing cell cycle arrest in G0/G1 phase.Additionally,Poly I ∶ C induced the apoptosis of endothelial progenitor cells in a dose-dependent manner.Caspase 8 and Caspase 9 inhibitors did not reduce Poly I ∶ C-induced apoptosis of endothelial progenitor cells.TNF-α had no effect on apoptosis of endothelial progenitor cells.IL-1β induced cell apoptosis in a dose-dependent manner.Moreover,when endothelial progenitor cells pre-treated with anti-IL-1R1,then re-stimulated with Poly I ∶ C,the cell apoptosis induced by Poly I ∶ C was decreased.Conclusions Poly I ∶ C at high concentrations inhibited endothelial progenitor cells proliferation by inducing cell cycle arrest in G0/G1 phase and inducing cell apoptosis of endothelial progenitor cells.Poly I ∶ C induced the apoptosis of endothelial progenitor cells through up-regulating the expression of IL-1β via activating TLR3.Endogenous and exogenous apoptosis pathway and TNF-α did not contribute to Poly I ∶ C-induced cell apoptosis.