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

摘要

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、500 μmol·L-1 Hcy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、500 μmol·L-1 Hcy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LD-LR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 Lhe characLerisLics and funelion of smooLh muscle cell LDLR DNA meLhylaLion caused by homocyslcinc. Methods 50, 100, 200, 500 μmol o L-1 llcy was addcd into Lhe primary cul-Lured VSMCs for 72 hours respectively. The promoLer DNA meLhylaLion sLaLus of LDLR was examined by nMS-PCR. The capaciLy of LDLR Lo accepL meLhyl groups was deLecLed by radioacLive isoLope and meLhyl-aLion-dependenL resLricLion analysis. Results The promoLer sLaus of LDLR was hypomeLhylaLion in 50, 100, 200, 500 μmol · L-1 llcy groups( P < 0. 05 ). With the digesLion by meLhyl-specific endonuclease llpall and BssIIII, the sequences CCGG and GCGCGC of LDLR promotcr were cleaved respectivcly. The meLhylaLion-accepling capaciLy was decreased and especially digesLed by llpall. Conclusion llcy causes LDLR DNA meLhylaLion siLe probably in Lhe C ↓ CGG sequence insLead of Lhe GC, ↓ GCATC.