ATP对人脐静脉内皮细胞增殖的抑制作用及其可能机制

ATP Inhibits the Proliferation of Human Umbilical Vein Endothelial Cell and Its Possible Mechanism

目的观察胞外核苷酸ATP对人脐静脉内皮细胞增殖活性及其生物学功能的影响,并初步探讨其可能机制。方法通过CCK-8细胞增殖试验检测不同浓度的ATP（0、1、5、10、50和100μmol/L）持续干预脐静脉内皮细胞3天及ATP（50μmol/L）不同时间段干预（1-6天）对脐静脉内皮细胞细胞增殖的影响;通过流式细胞术检测不同浓度的三磷酸腺苷诱导脐静脉内皮细胞凋亡情况及对其细胞生长周期的影响;通过RT-PCR对脐静脉内皮细胞表达的P2受体亚型进行检测,检测不同浓度的ATP（0、5、10、50和100μmol/L）对脐静脉内皮细胞表达P2Y2、P2Y11,细胞周期素cyclinB1、cyclinD1及细胞间粘附分子1、血管细胞粘附分子1和内皮型一氧化氮合酶的影响。结果与对照组比较,三磷酸腺苷在高浓度（50和100μmol/L）持续作用于脐静脉内皮细胞3天后显著抑制生长（P〈0.01）,终浓度50μmol/L的ATP呈时间依赖性抑制脐静脉内皮细胞生长。不同浓度的ATP对脐静脉内皮细胞凋亡均无明显影响,但在高浓度时可显著增加S期的细胞比例和减少G2/M期的细胞比例从而将细胞周期阻滞在S期;静止状态下,脐静脉内皮细胞表达P2X-4,5和P2Y-2,4,11,13,14;三磷酸腺苷各浓度组均能明显上调脐静脉内皮细胞细胞间粘附分子1和血管细胞粘附分子1的表达,仅在高浓度时上调P2Y2,11、eNOS的表达和下调cy-clinB1表达,而对表达cyclinD1的影响不明显。结论胞外核苷酸三磷酸腺苷在高浓度（50和100μmol/L）时可能通过受体P2Y2和P2Y11下调cyclinB1表达,使细胞周期从S期向G2/M期的转变受阻,从而抑制脐静脉内皮细胞生长,并且促进脐静脉内皮细胞表达动脉粥样硬化相关因子。

Aim To observe the effect of extracellular nucleotide ATP on proliferation activity and biological function of human umbilical vein endothelial cell（HUVEC）,and to explore its possible mechanism. Methods CCK-8 reagent kit was used to detect the effect of ATP intervening of different concentrations（0,1,5,10,50,100 μmol/L） and different time point（1~6 day） on proliferation of HUVEC;Flow cytometry was performed to detect apoptosis and cell cycle phase after treated with ATP as above;After treated with ATP of different concentrations（0,5,10,50,100 μmol/L） for 24 h,RT-PCR was performed to detect firstly the expression of P2 subtypes in HUVEC,secondly the expression of P2Y2,P2Y11,cyclinB1 and cyclinD1 and finally the expression of intercellular adhesion molecule（ICAM-1）,vascular cell adhesion molecule（VCAM-1）,endothelial nitric oxide synthase（eNOS）. Results Compared with the control group,ATP groups（50 and 100 μmol/L） significantly inhibited the growth of HUVEC（P0.01） and the inhibition of ATP（50 μmol/L） on HUVEC growth was time-dependent;ATP had no significant effect on HUVEC apoptosis,but high concentrations significantly increased the proportion of cells in S phase and diminished the proportion of cells in G2/M phase;ATP upregulated expression of ICAM-1,VCAM-1 in HUVEC in all concentration groups,and upregulated expression of P2Y2,11,eNOS and down-regulated expression of cyclinB1 only at high concentrations,while had no signifigant effect on cyclinD1 expression. Conclusion ATP at high concentrations（50,100 μmol/L） significantly inhibited HUVEC growth by blocking cell cycle in S phase and promoted expression of atherosclerosis-related adhesion molecules in HUVEC,which may be relevant to its role of up-regulating the expression of P2Y2,11 and down-regulating the expression of cyclinB1.