同型半胱氨酸引起平滑肌细胞LDLR启动子区DNA甲基化改变的位点分析

Characteristics and function of homocysteine-induced LDLR promoter DNA methylation in vascular smooth muscle cells

目的探讨同型半胱氨酸(Hcy)引起平滑肌细胞低密度脂蛋白受体(LDLR)启动子区DNA甲基化改变的特点及机制。方法原代培养人脐静脉平滑肌细胞(VSMCs)并鉴定,用50、100、200、500μmol·L-1Hcy干预,巢式降落式甲基化特异性PCR(nMS-PCR)法检测LDLR甲基化改变,甲基敏感性限制性内切酶HpaII和BssHII分析LDLR DNA甲基接受能力。结果 50、100、200、500μmol·L-1Hcy组平滑肌细胞LDLR启动子区DNA去甲基化(P<0.05),经限制性内切酶HpaII消化后,LDLR启动子区C↓CGG序列被酶切,经限制性内切酶BssHII消化后,LDLR启动子区GC↓GCGC序列被酶切,与对照组比较,DNA甲基接受能力下降,其中HpaII引起的效果更明显。结论 Hcy可使平滑肌细胞LDLR启动子区DNA去甲基化的效应偏重于一般的CpG二核苷酸序列,CpG岛所受影响相对较小。

Aim To explore the characteristics and function of smooth muscle cell LDLR DNA methylation caused by homocysteine.Methods 50,100,200,500 μmol·L-1 Hcy was added into the primary cultured VSMCs for 72 hours respectively.The promoter DNA methylation status of LDLR was examined by nMS-PCR.The capacity of LDLR to accept methy1 groups was detected by radioactive isotope and methylation-dependent restriction analysis.Results The promoter staus of LDLR was hypomethylation in 50,100,200,500 μmol·L-1 Hcy groups（P0.05）.With the digestion by methyl-specific endonuclease HpaII and BssHII,the sequences CCGG and GCGCGC of LDLR promoter were cleaved respectively.The methylation-accepting capacity was decreased and especially digested by HpaII.Conclusion Hcy causes LDLR DNA methylation site probably in the C↓CGG sequence instead of the GC↓GCGC.