CAJ | 학술논문

目的观察免疫相关性血细胞减少症(IRP)患者CD4+T细胞基因组DNA总体甲基化水平及甲基化相关调控基因的表达水平,探讨甲基化在IRP发病中的作用.方法选取2012年12月至2013年12月天津医科大学总医院血液科确诊的30例IRP患者(15例初治患者,15例恢复患者)及15名健康对照,以免疫磁珠法分选CD4+T细胞,应用ELISA法测定外周血CD4+T细胞DNA总体甲基化水平;采用反转录PCR (RT-PCR)技术检测CD4+T细胞中甲基化相关调控基因:DNA甲基化转移酶(DNMT)和甲基结合蛋白(MBD)的mRNA表达水平,并进行相关性分析.结果 IRP患者外周血CD4+T细胞DNA总体甲基化水平初治组和恢复组均明显低于对照组(3.525％±2.046％和4.790％±1.471％比10.101％±3.449％,均P＜0.05);初治组DNMT3b mRNA表达水平显著低于对照组(1.332±0.785比2.077±1.059,P＜0.05);初治组、恢复组MBD2 mRNA表达水平显著高于对照组(2.999±1.601和2.055±1.576比0.581±0.247,均P＜0.05);初治组MBD4 mRNA表达水平显著高于对照组(2.736±2.719比1.167±1.006,P＜0.05).IRP患者外周血CD4+T细胞DNMT3b mRNA表达水平与DNA总体甲基化水平呈正相关(r=0.569,P＜0.01);MBD2 mRNA表达水平与CD4+T细胞基因组DNA甲基化水平和Th1/Th2水平均呈负相关(r=-0.763,P＜0.01;r=-0.652,P＜0.05).CD4+T细胞甲基化水平与CD5+B细胞比例呈负相关(r=-0.439,P ＜0.05).结论 IRP患者外周血CD4+T细胞基因组DNA低甲基化和甲基化相关调控基因表达异常可能与IRP发病有关.
목적 관찰면역상관성혈세포감소증(IRP)환자CD4+T세포기인조DNA총체갑기화수평급갑기화상관조공기인적표체수평,탐토갑기화재IRP발병중적작용.방법 선취2012년12월지2013년12월천진의과대학총의원혈액과학진적30례IRP환자(15례초치환자,15례회복환자)급15명건강대조,이면역자주법분선CD4+T세포,응용ELISA법측정외주혈CD4+T세포DNA총체갑기화수평;채용반전록PCR (RT-PCR)기술검측CD4+T세포중갑기화상관조공기인:DNA갑기화전이매(DNMT)화갑기결합단백(MBD)적mRNA표체수평,병진행상관성분석.결과 IRP환자외주혈CD4+T세포DNA총체갑기화수평초치조화회복조균명현저우대조조(3.525％±2.046％화4.790％±1.471％비10.101％±3.449％,균P＜0.05);초치조DNMT3b mRNA표체수평현저저우대조조(1.332±0.785비2.077±1.059,P＜0.05);초치조、회복조MBD2 mRNA표체수평현저고우대조조(2.999±1.601화2.055±1.576비0.581±0.247,균P＜0.05);초치조MBD4 mRNA표체수평현저고우대조조(2.736±2.719비1.167±1.006,P＜0.05).IRP환자외주혈CD4+T세포DNMT3b mRNA표체수평여DNA총체갑기화수평정정상관(r=0.569,P＜0.01);MBD2 mRNA표체수평여CD4+T세포기인조DNA갑기화수평화Th1/Th2수평균정부상관(r=-0.763,P＜0.01;r=-0.652,P＜0.05).CD4+T세포갑기화수평여CD5+B세포비례정부상관(r=-0.439,P ＜0.05).결론 IRP환자외주혈CD4+T세포기인조DNA저갑기화화갑기화상관조공기인표체이상가능여IRP발병유관.
Objective To explore the global DNA methylation and the expression of regulatory genes for methylation in CD4 + T cells of the patients with immune related pancytopenia (IRP) and explore the role of methylation in the pathogenesis of IRP.Methods Thirty IRP patients (untreated,n =15 ;remission,n =15) and 15 healthy donors as controls were enrolled from December 2012 to December 2013.CD4 + T cells were sorted by immunomagnetic separation.The global DNA methylation was tested with enzyme-linked immunosorbent assay (ELISA).The mRNA levels of DNA methylation-related regulating genes,DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs),were measured by real-time quantitative-polymerase chain reaction (RT-PCR).Results The level of global DNA methylation in peripheral blood CD4 + T cells of untreated IRP patients (3.525％ ± 2.046％) and remission patients (4.790％ ± 1.471％)were significantly lower than that of normal controls (10.101％ ± 3.449％) respectively (both P ＜ 0.05).DNMT3b mRNA level of untreated IRP patients (1.332 ± 0.785) wassignificantly lower than that of normal controls (2.077 ± 1.059) in CD4 + T cells (P ＜ 0.05).The mRNA expression of MBD2 was significantly higher in CD4 + T cells from untreated and remission IRP patients (2.999 ± 1.601,2.055 ± 1.576) than that in controls(0.581 ±0.247) (both P ＜0.05).The MBD4 mRNA level was significantly higher in CD4 + T cells from untreated IRP patients (2.736 ± 2.719) compared to that in normal controls (1.167 ± 1.006) (P ＜ 0.05).DNMT3b mRNA expression and CD4 + T cell DNA methylation to be positive correlated within IRP patients (r =0.569,P ＜ 0.01).The MBD2 mRNA expression was negatively correlated with CD4 + T cell DNA methylation and the ratio of Th1/Th2 (r =-0.763,P ＜ 0.01; r =-0.652,P ＜ 0.05).The global methylation of CD4 + T cells negatively related to the ratio of CD5 + B cells (r =-0.439,P ＜ 0.05).Conclusion The globe DNA hypomethylation and abnormal expression of DNA methylation-related enzymes in peripheral blood CD4 + T cells may be related with the pathogenesis of IRP.