上海交通大学学报（医学版）

›› 2009, Vol. 29 ›› Issue (10): 1182-.

• 论著（基础研究） • 上一篇    下一篇

同型半胱氨酸诱导内皮祖细胞凋亡的氧化应激机制

鲍晓梅, 吴春芳, 陆国平   

  1. 上海交通大学 医学院瑞金医院心内科, 上海 200025
  • 出版日期:2009-10-25 发布日期:2009-10-26
  • 作者简介:鲍晓梅(1972—), 女, 博士生；电子信箱: baoxiaomei_nt@yahoo.com.cn。

Oxidative mechanism of homocysteine-induced apoptosis in endothelial progenitor cells

BAO Xiao-mei, WU Chun-fang, LU Guo-ping   

  1. Department of Cardiology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2009-10-25 Published:2009-10-26

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摘要：

目的 探讨同型半胱氨酸(Hcy)诱导小鼠骨髓内皮祖细胞（EPCs）凋亡的氧化应激机制。方法 密度梯度离心法获取小鼠骨髓单个核细胞，培养7 d后收集贴壁细胞，荧光显微镜下鉴定EPCs；与不同浓度Hcy(0、50、100、500 μmol/L)共孵育12、24、48 h，或分别经氧自由基清除剂NAC（1 mmol/L）、NADPH氧化酶抑制剂DPI（10 μmol/L）、p38MAPK特异性抑制剂SB203580（10 μmol/L）预孵育30 min后，再与500 μmol/L Hcy共孵育24 h；Annexin-V/ PI双染色经流式细胞术检测细胞凋亡，荧光探针H2DCF-DA法检测细胞内活性氧水平，光泽精化学发光法检测NADPH氧化酶活性，硝酸还原酶法测定细胞培养液中一氧化氮（NO）含量。结果 Hcy呈时间、剂量依赖性诱导EPCs凋亡、活性氧产生及NADPH氧化酶激活，并减少细胞培养液中NO含量（P<0.05或P<0.01）；NAC、DPI、SB203580预处理能拮抗Hcy诱导的上述改变（P<0.05或P<0.01）。结论 Hcy可能通过激活NADPH氧化酶、诱导EPCs活性氧的产生、降低NO水平及激活p38MAPK途径，导致EPCs凋亡。

关键词: 同型半胱氨酸, 内皮祖细胞, 凋亡, 活性氧, NADPH氧化酶, p38MAPK

Abstract:

Objective To investigate the oxidative mechanism of homocysteine (Hcy)-induced apoptosis in endothelial progenitor cells (EPCs). Methods Total mononuclear cells were isolated from mouse bone marrow by Ficoll density gradient centrifugation and were cultured in vitro for 7 d. Adherent cells were harvested and identified by fluorescence microscopy. EPCs were cultured with Hcy (0, 50, 100 and 500 μmol/L) for 12, 24 and 48 h, or pretreated with NAC(1 mmol/L), DPI(10 μmol/L) or SB203580 (10 μmol/L) for 30 min, then cultured with 500 μmol/L Hcy for 24 h. Apoptosis was detected by Annexin-V/PI flow cytometry, levels of reactive oxygen species (ROS) in cells were measured using H2DCF-DA as a fluorescence probe, NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence, and NO in the supernatant was determined by nitrate reductase assay. Results Hcy induced EPCs apoptosis, ROS accumulation, NADPH oxidase activation and decrease of NO in a time-dose dependent manner（P<0.05 or P<0.01）. Pretreatment with NAC, DPI and SB203580 could inhibit these effects （P<0.05 or P<0.01）. Conclusion Hcy could activate NADPH oxidase, induce ROS increase and NO decrease, and activate p38MAPK to enhance EPCs apoptosis.

Key words: homocysteine, endothelial progenitor cells, apoptosis, reactive oxygen species, NADPH oxidase, p38MAPK

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