基因组DNA甲基化在同型半胱氨酸致平滑肌细胞增殖的作用机制研究

The Mechanism of Genomic DNA Methylation in Hcy Induced VSMCs Proliferation

目的探讨基因组DNA甲基化变化在Hcy致VSMCs增殖的作用机制。方法原代培养VSMCs并鉴定,用0、50、100、200、500μM浓度Hcy干预,MTT法检测VSMCs活性;SssI甲基转移酶法分析基因组DNA甲基接受能力,甲基化敏感性限制性内切酶法分析基因组DNA甲基接受能力及DNA甲基转移酶活性。结果 MTT法表明在50μM Hcy时,VSMCs活性明显增加,其余活性下降,与各浓度组并不呈量效依赖关系;Hcy可增加DNA甲基接受能力,以50μM Hcy组效应最显著(P<0.05);HpalI限制核酸内切酶消化,与对照组比较,基因组DNA甲基化接受能力减少了50.1%、41.7%、46.1%和25.5%(50、100、200、500μM Hcy组),BssHII限制核酸内切酶消化后,基因组DNA甲基化接受能力减少了约25.5%、18.1%、17.4%和18.0%(50、100、200、500μM Hcy组)。结论 Hcy引起VSMCs基因组DNA去甲基化是平滑肌细胞增殖的重要机制之一,其效应偏重发生于CpG二核苷酸序列,CpG岛所受影响相对较小,其最大效应在50μMHcy组,且无明显的量-效依赖关系。

Objective To investigate the mechanism of Genomic DNA methylation in Hcy induced VSMCs proliferation.Methods 0,50,100,200,500μM concentrations of Hcy were added into the primary cultured VSMCs.Proliferative activity of VSMCs measured by MTT;genomic DNA methyl acceptance was analyzed with SssI methyltransferase enzyme method;genomic DNA methyl acceptance and Determination of DNA methyltransferase activety were determined with Methylation-sensitive restriction enzyme method;changes in genomic DNA methylation from multiple level were detected with repeated experiments.Results MTT test showed that 50mM Hcy increased the proliferation viability of VSMCs while 100,200 and 500 mM Hcy decreased the viable cells count;The impacts of various concentration of Hcy on the one-carbon methyl-group transferring metabolism didn＇t show dose-effect relationship.50mM Hcy could significantly increased genomic DNA methyl acceptance（P0.05）.In HpaII digestion group,its reductions in methylation-accepting capacity was 50.1%,41.7%,46.1% and 25.5%（50,100,200 and 500mM Hcy respectively） compared with the undigested DNA.The methylation-accepting capacity in BssHII cutting groups declined just approximately 25.5%,18.1%,17.4% and 18.0%（50,100,200 and 500mM Hcy respectively） compared with the undigested DNA.Conclusion Hcy could lead to genomic DNA hypomethylation in VSMCs and it is maybe an important mechanism for VSMCs proliferation.