摘要
目的:探讨S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)升高对血管内皮细胞的损伤效应与整体基因组甲基化的关系。方法:以永生化的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)为研究对象,用不同浓度的S-腺苷同型半胱氨酸水解酶(S-adenosylhomocysteine hydrolase,SAHH)抑制剂3-deazaadenosine(DZA)处理24、48、72h,使用光学显微镜观察细胞形态学变化,电子显微镜观察细胞器超微结构变化,反相高效液相色谱法(reversed phase high-performance liquid chromatography,RP-HPLC)测定胞内SAH浓度,荧光偏振免疫分析法(fluorescence polarization immunoassay,FPIA)测定Hcy的浓度,MTT法检测细胞生长增殖能力,流式细胞仪检测细胞凋亡率,胞嘧啶延伸法检测整体基因组DNA甲基化水平。结果:HUVEC经DZA处理后,形态学上出现凋亡或坏死特征,培养基中同型半胱氨酸(homocysteine,Hcy)浓度下降,胞内SAH含量升高,细胞的生长增殖能力降低并出现凋亡现象,整体基因组的甲基化水平降低。结论:SAH升高能导致血管内皮细胞的损伤,且这种作用与整体基因组甲基化水平有关,SAH可能是动脉粥样硬化形成过程中的潜在生物标志分子。
Objective： To explore the relationship of the damage induced by intracellular increased s-adenosylhomocysteine （SAH） and the whole DNA methylation in human umbilical vein endothelial cells （HUVEC）. Method.. After HUVEC were treated with different concentrations of potent s-adenosylhomo- cysteine hydrolase （SAHH） inhibitor 3-deazaadenosine （DZA） for 24, 48 and 72h, the cell morphology and cellular ultrastructure were observed with light microscope and eletron microscope respectively. The cellular SAH level was measured with reversed phase high-performance liquid chromatography （RP-HPLC）. The homocysteine （Hcy） concentration in medium was determined by fluorescence polarization immunoassay （FPIA）. The cell growth and proliferation were determined by MTT assay. The apoptotic percentage was analyzed with flow cytometry. The whole genome methylation was analyzed with cytosine extension method. Results. Compared to normal, the HUVEC after treated with DZA emerged the apoptosis and necrosis characters. DZA up-regulated the intracellular SAH level, down-regulated Hcy concentration in medium, and inhibited the celluar growth and proliferation. The level of whole genome methylation in DZA treated group was also lower than that of normal group. Conclusion. The intracellular increased SAH damages the HUVEC, which is correlated with whole DNA methylation. It＇s possible that SAH is the potential biological marker in atherogenesis.
出处
《营养学报》
CAS
CSCD
北大核心
2007年第6期547-551,共5页
Acta Nutrimenta Sinica
基金
国家自然科学基金(No.30571568)
中国博士后科学基金(No.2005037174)
