【作者】 苏娟； 王生兰； 黄玉珊； 姜怡邓； 王树人；

【Author】 SU Juan,WANG Shenglan,HUNAG Yushan,JINAG Yideng,et al. Department of Pathophysiology,West China School of Preclinical Medical Sciences & Forensic Medicine, Sichuan University,Chengdu 610041,China

【机构】 四川大学华西基础医学和法医学院病理生理学教研室；

【摘要】 目的以动脉粥样硬化(As)病灶中的主要细胞类型一血管平滑肌细胞为模型,对比研究同型半胱氨酸(Hcy)和半胱氨酸(Cys)在致As发病效应上的异同,以期为Hcy作为As独立危验因子的分子机制提供有比较的证据。方法体外培养人脐静脉血管平滑肌细胞(VSMCs),用不同浓度的Hcy和Cys作用于细胞24h后:(1)用分光光度比色法测定细胞中SOD和MDA的含量;(2)流式细胞术检测细胞凋亡率,半定量RTPCR方法检测caspases-3 mRNA的表达;(3)MTT法测定细胞的增殖率;(4)用1000μmol/L Hcy和Cys分别作用VSVCs 24、48和72h后,MS-PCR法检测ERα启动子区甲基化的状态,半定量RT-PCR法检测ERαmRNA的表达。结果 (1)Hcy可导致细胞内MDA、SOD呈剂量依赖性增加,Cys使MDA、SOD增加的效应远弱于Hcy(P<0) 01)。(2)流式细胞术、caspases-3 mRNA的表达和MTT试验的结果显示,Hcy、Cys对血管平滑肌细胞凋亡率和增殖率的影响差异不太明显。(3)Hcy显著增加ERα基因启动区的甲基化修饰,并使ERαmRNA的表达进行性减少。Cys不影响ERα启动子区的甲基化状态,对ERαmRNA表达的影响也远小于Hcy。结论氧化应激对DNA甲基化修饰的影响可能在Hcy致As发病的机制中占据着重要的地位,这也可能是Hcy和Cys在As发病机制中作用不同的重要原因。更多还原

【Abstract】 Objective To investigate the similarities and differences of homocysteine and cysteine on pathogenesis of As in cultured human vascular smooth muscle cells(HVSMCs).Methods Cultured HVSMCs were treated by different concentrations of Hcy and Cys for 24 h.(1) The SOD activity and MDA contents were measured by chromatometry.(2) The rates of apoptosis of HVSMCs were detected by flow cytometry.The expression of caspases-3 mRNA in HVSMCs was detected by reverse-trascription polymerase chain reaction(RT-PCR).(3) The proliferative activities of HVSMCs were examined by MTT assay.(4) HVSMCs were incubated with 1000μmol/L Hcy and Cys for 24,48 and 72h respectively, then the DNA methylation status were assayed by nested methylation-specific polymerase chain reaction,and the expressions of ERαmRNA of HVSMCs were detected by RT-PCR.Results(1) Hcy significantly increased the MDA contents and SOD activities in a dose-dependent manner,but the increase of MDA contents and SOD activities were much weaker induced by Cys(P<0.01).(2) The results of flow cytometry,expression of caspases-3 mRNA,and MTT test showed that the effects of Hcy and Cys on rate of apoptosis and proliferation of HVSMCs were not much different.(3) Hcy obviously induced de novo methylation in the promoter region of the ERαgene in HVSMCs,and the expressions of ERαmRNA were correspondingly down-regulated.The Cys had no effect on methylation modification of ERαgene,and very mild effect on ERαmRNA expression.Conclusion The oxidative stress and the methylative modification induced by Hcy may have the top position in its pathogenic mechanism of pro-atherosclerosis.The pro-apoptotic and proliferation-promoting effects of Hcy were similar with Cys,which suggested that these two effects might just play mild roles in its proatheroscleroatic traits.更多还原