冠心病患者CD14^+单核细胞总体DNA异常甲基化及其机制研究

Research of aberrant global DNA methylation level in CD14^+ monocytes in patients with coronary artery disease and its mechanism

目的探讨冠心病患者外周血CD14^+单核细胞基因组总体DNA异常甲基化及其机制。方法密度梯度离心法和磁珠分选法分离试验组(冠心病患者)和对照组(非冠心病患者)外周血CD14^+单核细胞。用基因组总体甲基化定量试剂盒检测DNA总体甲基化水平;用实时荧光定量聚合酶链式反应(qRT-PCR)检测DNA甲基转移酶(DNMTs)和甲基结合蛋白2(MBD2)的mRNA表达水平。结果对照组与试验组CD14^+单核细胞DNA总体甲基化水平分别为0.30±0.04,0.44±0.03,差异有统计学意义(P<0.05)。对照组与试验组DNMT1 mRNA表达水平分别为2.16±0.32,1.40±0.13,差异有统计学意义(P<0.05);DNMT3A mRNA表达水平分别为0.56±0.05,0.89±0.12,差异有统计学意义(P<0.05);DNMT3B mRNA表达水平分别为0.59±0.07,0.97±0.10,差异有统计学意义(P<0.01);MBD2 mRNA表达水平分别为1.68±0.20,1.20±0.08,差异有统计学意义(P<0.05)。冠心病试验组中,CD14^+单核细胞DNMT1的mRNA表达水平与DNA总体甲基化水平呈明显正相关(r=0.648),差异有统计学意义(P<0.05);DNMT3B的mRNA表达水平与DNA总体甲基化水平呈明显正相关(r=0.700),差异有统计学意义(P<0.05);MBD2的mRNA表达水平与DNA总体甲基化水平呈显著负相关(r=-0.540),差异有统计学意义(P<0.05),而表达升高的DNMT3A与总体甲基化水平没有明显相关性(P>0.05)。结论冠心病患者外周血CD14^+单核细胞基因组DNA呈总体高甲基化,可能是DNMT1、DNMT3A和DNMT3B和MBD2共同作用的结果。

Objective To research mainly focuses on the changes of genomic DNA global methylation of peripheral blood CD14~＋ monocytes in patients with coronary artery disease（ CAD）. Methods CD14~＋ monocytes were isolated from peripheral blood of treatment group（ CAD patients） and control group（ no-CAD patients） by density gradient centrifugation and magnetic bead selection. We detected genomic DNA global methylation level with genome global methylation quantitation kit.Quantitive real-time polymerase chain reaction（ qRT-PCR） was performed to detect the expression levels of DNA methyltransferases（ DNMTs） and methyl binding protein 2（ MBD2） mRNA. Results In control group and treatment group,genomic DNA global methylation levelsof CD14~＋ monocytes were 0. 30 ± 0. 04,0. 44 ± 0. 03,the difference was statistically significant（ P〈0. 05）. In control group and treatment group,the expression levels of DNMT1 mRNA were 2. 16 ± 0. 32,1. 40 ± 0. 13,with significant difference（ P〈0. 05）. The expression levels of DNMT3 A mRNA were 0. 56 ± 0. 05,0. 89 ± 0. 12,with significant difference（ P〈0. 05）. The expression levels of DNMT3 B mRNA were 0. 59 ± 0. 07,0. 97 ± 0. 10,with significant difference（ P〈0. 01）. The expression levels of MBD2 mRNA were 1. 68 ± 0. 20,1. 20 ± 0. 08,with significant difference（ P〈0. 01）. In treatment group of coronary heart disease,global methylation level and the expression levels of DNMT1 mRNA（ r = 0. 648） as well as DNMT3B（ r = 0. 700） were positive correlated and statistically significant（ both P〈0. 05）,global methylation level and the expression levels of MBD2（ r =-0. 540） were negative correlated and statistically significant（ P〈0. 05）,while global methylation level and the expression level of DNMT3A were not correlated,the difference was not significant（ P〉0. 05）. Conclusion Genomic DNA of peripheral CD14~＋ monocytes in patients with coronary artery disease presents global hypermethylation,which may be as a result of out-of-balance between methylation and demethylation induced by abnormal expressions of DNMTs and MBD2.