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目的以动脉粥样硬化(As)病灶中的主要细胞类型—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RT-PCR方法检测caspases-3 mRNA的表达;(3)MTT法测定细胞的增殖率;(4)用1000μmol/LHcy和Cys分别作用VSMCs24、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ERα mRNA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ERα mRNA的表达进行性减少。Cys不影响ERα启动子区的甲基化状态,对ERα mRNA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。
Objective To compare the similarities and differences between homocysteine (Hcy) and cysteine (Cys) in the pathogenesis of As with the model of vascular smooth muscle cells, the main cell type in atherosclerotic lesions. Provide comparative evidence for the molecular mechanism of Hcy as an independent risk factor for As. Methods Human umbilical vein smooth muscle cells (VSMCs) were cultured in vitro and treated with different concentrations of Hcy and Cys for 24 hours. (1) The contents of SOD and MDA in the cells were measured by spectrophotometry. (2) Flow cytometry (3) MTT method was used to determine the proliferation rate of cells; (4) VSMCs were treated with 1000μmol / L LHcy and Cys for 24, 48 and 72 hours, respectively. The methylation status of ERα promoter region was detected by MS-PCR and the expression of ERα mRNA was detected by semi-quantitative RT-PCR. Results (1) Hcy could induce a dose-dependent increase of MDA and SOD in the cells, and the effect of Cys on the increase of MDA and SOD was much weaker than that of Hcy (P <0.01). (2) Flow cytometry, caspases-3 mRNA expression and MTT test results showed that Hcy, Cys on vascular smooth muscle cell apoptosis rate and proliferation rate difference is not obvious. (3) Hcy significantly increased the methylation of the promoter region of ERα gene and decreased the expression of ERα mRNA. Cys did not affect the methylation status of ERα promoter, and its effect on ERα mRNA expression was much less than that of Hcy. Conclusion The effect of oxidative stress on DNA methylation may play an important role in the pathogenesis of As induced by Hcy, which may be an important reason for the different roles of Hcy and Cys in As pathogenesis.