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mRNA Expression of Methylase and Methyl-CpG Binding Protein in Peripheral Blood Mononuclear Cells from Patients with Generalized Pustular Psoriasis
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摘要:
目的 观察泛发性脓疱型银屑病(generalized pustular psoriasis，GPP)患者外周血单个核细胞(peripheral blood mononuclear cell，PBMC)中DNA甲基转移酶及甲基化CpG结合蛋白(methyl-CpG binding protein，MeCP)mRNA的表达情况，初步探讨DNA甲基化在GPP中可能的作用机制。 方法 回顾性收集2015年12月至2016年12月在北京协和医院皮肤科门诊或住院治疗的9例GPP患者临床资料；随机选取10例同期在医院接受健康体检的健康人做为对照，年龄和性别与GPP患者相匹配。分别留取10 ml肘正中静脉血，采用密度梯度离心法分离PBMC，应用Trizol法提取PBMC总RNA，采用实时PCR法检测DNMT1、DNMT3a、DNMT3b、MBD1、MBD2、MBD3、MBD4及MeCP2的mRNA表达水平，并分析年龄及病情严重程度与mRNA表达水平的相关性。 结果 GPP患者PBMC中DNMT3a、DNMT3b、MBD1、MBD2、MBD4的mRNA表达水平较健康对照组升高[DNMT3a:0.000 17(0.000 06, 0.001 15)比0.000 03(0.000 02，0.000 04)；DNMT3b：0.000 04±0.000 02比0.000 02±0.000 01；MBD1：为0.001 01±0.000 45比0.000 46±0.000 15；MBD2:0.002 61±0.000 39比0.001 85±0.000 52；MBD4：0.004 29±0.001 60比0.001 57±0.000 55, P均＜0.05], 年龄及疾病严重度与上述因子mRNA的相对表达水平无相关性(P均＞0.05)。 结论 GPP患者体内甲基化相关调控基因表达异常，但与年龄和疾病严重程度不相关。 -
关键词:
- DNA甲基转移酶 /
- 甲基化CpG结合蛋白 /
- 泛发性脓疱型银屑病
Abstract:Objective The aim of this study was to observe the mRNA expression of DNA methyl-transferase(DNMT) and methyl-CpG binding protein(MeCP) in the peripheral blood mononuclear cells from GPP patients, and to explore the potential role of DNA methylation in the pathogenesis of GPP. Methods Clinical data of 9 GPP patients admitted or hospitalized at the Department of Dermatology of Peking Union Medical College Hospital from December 2015 to December 2016 were retrospectively collected. Ten healthy people who underwent a physical examination in the same hospital during the same period were randomly selected as the control group; their age and gender were matched with those of GPP patients. Peripheral blood mononuclear cells were isolated from 10 ml blood of the elbow median vein using density gradient centrifugation. Total RNA was extracted from peripheral blood mononuclear cells by Trizol method; mRNA expression levels of DNMT1, DNMT3a, DNMT3b, MBD1, MBD2, MBD3, MBD4, and MeCP2 were detected by real-time PCR. The correlation of age and GPP severity with the expression level of these mRNAs was observed and analyzed. Results Compared with the healthy subjects, the relative expressions of DNMT3a, DNMT3b, MBD1, MBD2, and MBD4 mRNA in peripheral blood mononuclear cells from GPP patients were higher than those from the healthy controls [DNMT3a:0.000 17(0.000 06, 0.001 15) vs. 0.000 03(0.000 02, 0.000 04), DNMT3b 0.000 04± 0.000 02 vs. 0.000 02±0.000 01, MBD1 0.001 01±0.000 45 vs. 0.000 46±0.000 15, MBD2 0.002 61±0.000 39 vs. 0.001 85±0.000 52; and MBD4 0.004 29±0.001 60 vs. 0.001 57±0.000 55, all P < 0.05]. There was no correlation between age and GPP severity with the mRNA expression of the above factors (all P > 0.05). Conclusions Methylation related regulatory genes are abnormally expressed in GPP patients, and do not correlate with age or the disease severity. 利益冲突 无 -
表 1 GPP患者甲基化相关调控基因引物序列
基因名称 上游引物(5′→3′) 下游引物(5′→3′) DNMT1 5′-GAGCTACCACGCAGACATCA-3′ CGAGGAAGTAGAAGCGGTTG DNMT3a 5′-CCGGAACATTGAGGACATCT-3′ CAGCAGATGGTGCAGTAGGA DNMT3b 5′-CCCATTCGAGTCCTGTCATT-3′ GGTTCCAACAGCAATGGACT MBD1 5′-CACCCTCTTCGACTTCAAACAAG-3′ CAACCTGACGTTTCCGAGTCTT MBD2 5′-AACCCTGCTGTTTGGCTTAAC-3′ CGTACTTGCTGTACTCGCTCTTC MBD3 5′-CCGCTCTCCTTCAGTAAATGTAAC-3′ GGCTGGAGTTTGGTTTTCAGAA MBD4 5′-TGGTGGTGCATGCCTGTAAT-3′ TGAGACAGGGTCTCTCTCTGTCAT MeCP2 5′-CCCCACCCTGCCTGAA-3′ GATGTGTCGCCTACCTTTTCG GPP:泛发性脓疱型银屑病；DNMT：DNA甲基转移酶；MBD：甲基化CpG结合域; MeCP:甲基化CpG结合蛋白 
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表 2 GPP患者及健康人外周血单个核细胞中DNMT和MeCP mRNA相对表达量的比较
基因 GPP组(n=9) 健康对照组(n=10) t/Z值 P值 DNMT1 0.001 77±0.000 76 0.001 83±0.002 00 -0.097 0.924 DNMT3a 0.000 17
(0.000 06, 0.001 15)0.000 03
(0.000 02, 0.000 04)-2.598 0.009 DNMT3b 0.000 04±0.000 02 0.000 02±0.000 01 2.326 0.021 MeCP2 0.002 08±0.000 84 0.001 79±0.000 83 0.716 0.487 MBD1 0.001 01±0.000 45 0.000 46±0.000 15 3.060 0.000 MBD2 0.002 61±0.000 39 0.001 85±0.000 52 3.284 0.005 MBD3 0.000 02±0.000 01 0.000 03±0.000 01 -1.828 0.088 MBD4 0.004 29±0.001 60 0.001 57±0.000 55 4.447 0.003 GPP、DNMT、MBD、MeCP：同表 1
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表 3 GPP患者年龄和疾病严重程度与DNMT及MeCP mRNA表达水平的相关分析
风险因素 基因 r值 P值 年龄 DNMT1 -0.319 0.402 DNMT3a -0.067 0.864 DNMT3b -0.487 0.183 MBD1 -0.235 0.542 MBD2 -0.252 0.513 MBD3 -0.328 0.389 MBD4 -0.202 0.603 MeCP2 0.050 0.897 病情严重程度 DNMT1 -0.134 0.732 DNMT3a 0.098 0.802 DNMT3b -0.241 0.533 MBD1 -0.241 0.533 MBD2 -0.009 0.982 MBD3 -0.178 0.646 MBD4 -0.134 0.732 MeCP2 -0.285 0.457 GPP、DNMT、MBD、MeCP：同表 1
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