在载脂蛋白E基因敲除小鼠和人脐静脉平滑肌细胞中同型半胱氨酸对B1和Alu重复序列甲基化的影响  

Effect of Homocysteine on the Methylation Status of B1 and Alu Repetitive Sequences in ApoE^(-/-) Mice and Human Vascular Smooth Muscle Cells

作　　者：吴凯[1] 马胜超[2] 孙炜炜[3] 王菊[3] 曹成建[3] 马长剑[3] 杨安宁[1] 姜怡邓[1,4] 

机构地区：[1]宁夏医科大学基础医学院,宁夏银川市750004 [2]四川大学华西基础与法医学院,四川省成都市610041 [3]宁夏医科大学检验学院,宁夏银川市750004 [4]宁夏医科大学总医院,宁夏银川市750004

出　　处：《中国动脉硬化杂志》2013年第9期785-790,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金(81260105;81160044和81260063);2010年教育部新世纪优秀人才支持计划(NCET-100916);宁夏自然科学基金项目(NZ11176);宁夏自治区科技厅科技攻关项目(20100820;[2012]17)

摘　　要：目的观察同型半胱氨酸对载脂蛋白E基因敲除小鼠不同组织和人脐静脉平滑肌细胞中B1和Alu序列甲基化水平的影响,为进一步阐明同型半胱氨酸致动脉粥样硬化形成的分子机制提供理论和实验依据。方法复制高同型半胱氨酸血症载脂蛋白E基因敲除小鼠模型,给予不同饮食14周后,分别取小鼠心脏组织、血管及白细胞;原代培养人脐静脉平滑肌细胞,用50、100、200及500μmol/L同型半胱氨酸干预培养72 h后,抽提组织、白细胞及平滑肌细胞的基因组DNA,采用巢式降落式甲基化特异性PCR法分别检测不同组织和细胞中B1和Alu序列甲基化水平的变化。结果饲以高蛋氨酸饮食的载脂蛋白E基因敲除小鼠心脏组织、血管和白细胞中B1重复序列甲基化水平下降明显,与正常对照组比较有显著性差异(P<0.05和P<0.01);在同型半胱氨酸干预的平滑肌细胞中Alu序列甲基化水平下降显著(P<0.05和P<0.01)。结论同型半胱氨酸引起B1和Alu序列低甲基化改变可能是其导致动脉粥样硬化发生发展的重要机制之一。Aim To observe the methylation levels of B1 and Alu sequences in different tissues of ApoE -/- mice and human vascular smooth muscle cells induced by homocysteine （Hcy）, so as to further clarify the molecular mechanisms of Hcy-induced atherosclerosis （As）. Methods The hyperhomocysteinemia ApoE-/- mice model were replicated, and the heart tissues, aortas and white blood ceils were taken from mice after fed for 14 weeks. 50, 100, 200, 500 p.mol/L Hcy were added into the primary cultured human umbilical vein smooth muscle cells for 72 h. The genomic DNA were extracted from the heart tissues, aortas, white blood cells and vascular smooth muscle cells. Then the methylation status of B1 and Alu repetitive sequences were examined by nMS-PCR. Results The methylation levels of B1 repeti- tive sequences were significantly decreased in the heart tissues, aortas and white blood cells of ApoE -/- mice fed with high methionine diet. Compared with the control group, there were significant differences （ P 〈 0. 05 and P 〈 0. 01 ）. The m-ethylation levels of Alu sequences in the smooth muscle cells cultured with Hcy were significantly declined （ P 〈 0. 05 and P 〈0. 01 ）. Conclusion The hypomethylation of B1 and Alu repetitive sequences induced by Hcy may be an important mechanism of As.

关 键 词：B1和Alu重复序列 甲基化 同型半胱氨酸 载脂蛋白E基因敲除小鼠 血管平滑肌细胞 

分 类 号：R363[医药卫生—病理学]