同型半胱氨酸对内皮细胞Bcl-2基因甲基化水平的影响

Effects of Homocysteine on Bcl-2 Gene Methylation of Endothelial Cell

目的研究同型半胱氨酸对人脐静脉内皮细胞Bcl-2基因启动子区甲基化水平及其表达的影响。方法以人脐静脉内皮细胞为实验对象,用含有不同浓度同型半胱氨酸的RPMI1640培养液对其作用48 h,MTT法测定细胞增殖生长能力并确定实验药物浓度;流式细胞术测定细胞凋亡水平;荧光定量PCR测定Bcl-2 mRNA表达;甲基化特异性PCR联合巢式降落式PCR测定Bcl-2启动子区甲基化水平。结果同型半胱氨酸对人脐静脉内皮细胞的增殖能力有明显的抑制作用,OD值从0.99±0.05逐渐下降至0.28±0.03(P<0.01),呈明显的剂量效应关系;与对照组相比,实验组细胞凋亡率由2.30%±0.60%上升至16.40%±0.73%(P<0.01),Bcl-2 mRNA表达明显下降(P<0.01),Bcl-2基因启动子区甲基化水平降低(P<0.05)。结论同型半胱氨酸通过下调Bcl-2启动子区的甲基化水平导致其mRNA表达水平减少,继而诱发内皮细胞凋亡,这可能在同型半胱氨酸促动脉粥样硬化过程中起到重要作用。

Aim To study the effects of homocysteine （Hcy） on methylation of Bcl-2 in human umbilical endothelial cells （HUVEC）. Methods After human umbilical endothelial cells were treated with different concentrations of Hcy for 48 h, MTT was used to measure the reproduction of human umbilical endothelial cells and IC50 concentration of Hcy, the flow cytometry assay was used to detect the rate of cell apoptosis, the expressions of Bcl-2 mRNA level were analyzed by fluorescence quantitative PCR, the methylation of Bcl-2 promoter region methylation level was determined by nest touch-down PCR combined methylation specific PCR （MSP）. Results The proliferation of human umbilical endothelial cells was inhibited by Hcy in a dose-independent manner, the OD value decreased from 0.99±0.05 to 0.28±0.03（P〈0.01）. Compared with control group, the rate of apoptosis in human umbilical endothelial cells increased from 2.30%±0.60% to 16.40%±0.73% （P〈0.01）, the expressions of Bcl-2 mRNA were significantly depressed （P〈0.01）, the Bcl-2 promoter region methylation levels in experimental group were significantly reduced （P〈0.05）. Conclusions Increasing endothelial cell apoptosis by decreasing Bcl-2 promoter region methylation level and reducing expression of Bcl-2 may play an important role in the pathogenesis of Hcy induced atherosclerosis.