内皮型一氧化氮合酶基因启动子区DNA甲基化调控同型半胱氨酸致内皮细胞损伤的机制  

The Mechanism of Endothelial Nitric Oxide Synthase Promoter DNA Methylation in the Impairment of Endothelial Cells Induced by Homocysteine

作　　者：姜怡邓[1] 孙炜炜[1] 马胜超[1] 王菊[1] 巩慧慧[1] 马长剑[1] 

机构地区：[1]宁夏医科大学检验学院,宁夏银川市750004

出　　处：《中国动脉硬化杂志》2013年第2期115-119,共5页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(30960124和81160044);教育部新世纪优秀人才支持计划(NCET-10-0916)

摘　　要：目的探讨内皮型一氧化氮合酶基因启动子区DNA甲基化在同型半胱氨酸致内皮细胞损伤中的作用机制。方法原代培养人脐静脉内皮细胞,加入0、50、100、200、500μmol/L同型半胱氨酸和100μmol/L同型半胱氨酸+维生素B12+叶酸的培养液中孵育72 h。MTT法检测人脐静脉内皮细胞的增殖活性;实时定量PCR测定内皮型一氧化氮合酶mRNA表达;化学比色法测定内皮型一氧化氮合酶活性;硝酸还原酶法检测一氧化氮生成量。巢式降落式甲基化特异性PCR法检测内皮型一氧化氮合酶基因启动子区DNA甲基化改变;同位素法检测DNA甲基化转移酶的活性。结果人脐静脉内皮细胞与不同浓度同型半胱氨酸孵育72 h后,人脐静脉内皮细胞的增殖活性降低,内皮型一氧化氮合酶mRNA表达、内皮型一氧化氮合酶活性和一氧化氮的含量明显下降。内皮型一氧化氮合酶基因启动子序列DNA甲基化程度随同型半胱氨酸浓度的升高而增加,且DNA甲基化转移酶活性升高,而叶酸和维生素B12有一定拮抗作用,与对照组比较差异有显著性(P<0.05和P<0.01)。结论同型半胱氨酸可导致内皮型一氧化氮合酶基因启动子区序列高甲基化修饰,并出现相应的内皮型一氧化氮合酶基因表达下调,这可能是同型半胱氨酸致内皮细胞损伤的重要机制之一。Aim To investigate the mechanism of endothelial nitric oxide synthase （eNOS） promoter DNA methylation in the damage of endothelial cells induced by homocysteine （Hey）. Methods Human umbilical vein endothelial cells （HUVEC） were cultured with Hcy at different concentrations （0, 50, 100,200 and 500 μmol/L） and 100 μmol/L Hcy ＋ Vitamin B12 ＋ folate respectively for 72 hours. The cell viability of HUVEC was detected by MTT, the mRNA expression of eNOS was analyzed by real-time PCR. The activity of eNOS was analyzed by ELISA, and the production of nitric oxide （NO） was examined by a commercial kit. Nested methylation specific polymerase chain reaction （nMS-PCR） was used for analysis of the methylation pattern in promoter regions of eNOS gene. The DNA methyhransferase activity was measured by isotope method. Results The viability of HUVEC exposed to different concentrations of Hcy decreased significantly. The mRNA levels of eNOS, eNOS activity and the NO production were significantly decreased. The methylation levels of eNOS promoter were Hcy-dose dependently increased, and the DNA methyltransferase activity was correlative increased. Conclusion Homocysteine can induce DNA hypermethylation of eNOS promoter, and the eNOS gene expression displayed correlative decrease, which may be an important mechanism for homocysteine-mediated endothelial impairment.

关 键 词：同型半胱氨酸 内皮型一氧化氮合酶 DNA甲基化 

分 类 号：R363[医药卫生—病理学]