过氧化氢诱导人脐静脉内皮细胞凋亡时核仁素的表达及细胞定位变化

Changes of nucleolin expression and cellular localization during HUVEC apoptosis induced by hydrogen peroxide

目的:探讨核仁素C23在过氧化氢(hydrogen peroxide,H2O2)致人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)凋亡中的表达及定位情况。方法:采用H2O2(0.5mmol/L)作用于人HUVEC,采用流式细胞术以及caspase-3活性检测观察细胞凋亡的发生情况,并通过逆转录-聚合酶链反应(RT-PCR)和Western印迹分别检测核仁素mRNA和蛋白质水平;间接免疫荧光检测核仁素的细胞内分布情况。结果:流式细胞术以及caspase-3活性检测均表明H2O2能明显地诱导HUVEC凋亡,并且Western印迹分析显示H2O2能够使核仁素发生断裂,RT-PCR分析显示H2O2诱导核仁素mRNA表达下调;间接免疫荧光检测显示核仁素从胞核移位至胞浆。结论:H2O2可诱导HUVEC凋亡,同时伴有核仁素表达下调及从胞核移位至胞浆。

Objective To investigate the expression and cellular localization of nucleolin C23 during human umbilical vein endothelial cell（HUVEC）apoptosis induced by hydrogen peroxide（H2O2）.Methods Apoptosis of HUVEC was induced by exposure to 0.5 mmol/L H2O2 for different periods and detected by flow cytometry and activity of caspase-3.The mRNA and protein expression of nucleolin were detected by reverse transcription-polymerase chain reaction（RT-PCR）and Western blot,respectively.The intracellular distribution of nucleolin was observed by indirect immunofluorescence.Results The percentage of apoptotic cells was increased significantly after treatment with H2O2 for 12,24 and 36 hours.The activity of caspase-3 reached the peak after treatment with H2O2 for 4 h.RT-PCR showed that nucleolin C23 mRNA was decreased after 2,4,and 8 hours treatment with H2O2.Western blot showed that C23 protein level was decreased after 12 hours with an additional cleft band of 80 kD appeared after 8 hours.Density analysis showed that the 80 kD cleft band increased in a time-dependent manner.Immunofluorescence analysis demonstrated that H2O2-induced C23 redistribution from the nucleus to the cytoplasm.Conclusion H2O2 could induce apoptosis accompanying with C23-cleavage and C23-translocation from the nucleus to the cytoplasm.