克拉屈滨对人脐静脉内皮细胞活力和代谢的影响

Effect of cladribine on growth inhibition and autocrining cytokines in human umbilical vein endothelial cell line EA. hy926

目的:研究克拉屈滨对人脐静脉内皮细胞EA.hy926生长及分泌活性的影响,探讨克拉屈滨通过抑制内皮细胞活力发挥抗肿瘤的作用机制。方法:采用CCK-8法检测不同浓度(0.4~1μmol/L)克拉屈滨对内皮细胞活力的影响;用流式细胞术检测细胞周期和细胞凋亡率;Western blot检测细胞凋亡和周期相关蛋白的表达水平;用ELISA法检测上清液肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、转化生长因子β1(transforming growth factor-β1,TGF-β1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的水平;Gries法检测上清液一氧化氮(nitric oxide,NO)含量。结果:克拉屈滨作用48 h对细胞活力有明显的抑制作用,其半数抑制浓度约为3.644μmol/L,随药物浓度升高和作用时间的延长而抑制作用增强。克拉屈滨作用48 h后,细胞周期被明显阻滞在S期,0.4μmol/L时S期细胞约43.74%,1μmol/L时S期细胞约77.23%。克拉屈滨处理后,各组内皮细胞的凋亡率没有显著差异。克拉屈滨处理后,caspase-3与Bax蛋白水平无明显差异;p21水平随着药物浓度增加而增高,而p53水平随着药物浓度增加而降低(P<0.05)。克拉屈滨处理后,上清液中TGF-β1和TNF-α水平升高,VEGF含量减少(P<0.05)。克拉屈滨处理后,上清液中NO含量减少(P<0.05)。结论:克拉屈滨能明显抑制内皮细胞EA.hy926的活力,其主要机制与细胞周期阻滞有关。同时克拉屈滨能促进内皮细胞EA.hy926分泌TNF-α和TGF-β1,抑制其分泌VEGF和NO。

AIM:To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926,and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells.METHODS:The effects of cladribine at different concentrations on the cell viability were detected by CCK-8 assay.Apoptosis and cell cycle distribution were examined by flow cytometry.The protein expression levels were determined by Western blot.The levels of tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA.The nitric oxide(NO)production was measured by Gries method.RESULTS:Cladribine at 0.4~1μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners.The IC 50 was about 3.644μmol/L.The results showed 43.74%cells in S phase when the concentration of cladribine was 0.4μmol/L,and 77.23%cells in S phase when the concentration of cladribine was 1μmol/L.The apoptosis was not induced by cladribine at 0.4~10μmol/L.The protein expression of Bax and caspase-3 did not change.The expression of p21 increased and the p53 decreased(P<0.05).The levels of TNF-αand TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h.The levels of VEGF and NO decreased.CONCLUSION:Cladribine obviously inhibits the viability of EA.hy926 cells.The mechanism is related to the cell cycle arrest.Cladribine promotes the secretion of TNF-αand TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.