心脏成纤维细胞分化可能与DNA甲基化修饰调控有关  

Modification of cardiac fibroblast differentiation by DNA methylation

作　　者：潘啸东[1] 何砚如 陈中璞[1] 刘玉[1] 马根山[1] 

机构地区：[1]东南大学附属中大医院心血管科,江苏南京210009

出　　处：《南京医科大学学报（自然科学版）》2016年第9期1052-1056,1062,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金青年项目(81500204);江苏省自然科学基金青年项目(BK20150648);东南大学基础科研扶持项目(3290005401)

摘　　要：目的:以转化生长因子-β1(transforming growth factorβ1,TGF-β1)体外诱导心脏成纤维细胞分化,探讨其中DNA甲基化的调控作用。方法:分离培养SD大鼠心脏成纤维细胞并行免疫细胞化学鉴定。取P1代细胞,分别以TGF-β1、DNA甲基转移酶(DNA methyltransferases,DNMT)抑制剂5-氮杂-2-脱氧胞苷(5-aza-2′-deoxycytidine,5-aza-d C)及TGF-β中和抗体进行干预。Western blot及细胞免疫荧光检测α平滑肌动蛋白(α-smooth muscle actin,α-SMA)的表达,实时定量PCR检测α-SMA以及DNMT1、DNMT3a、DNMT3b的基因表达,并进行DNMT活性测定。结果 :与对照组相比,TGF-β1与5-aza-d C一样均可诱导心脏成纤维细胞表达α-SMA,且TGF-β1可显著抑制总DNMT的活性(P<0.05),并可抑制DNMT1及DNMT3a的表达(P<0.01),但对DNMT3b的表达无明显影响(P>0.05)。结论:心脏成纤维细胞分化可能与DNA甲基化修饰调控有关,本研究从表观遗传学角度为心肌纤维化防治研究提供了新的治疗靶点。Objective： To explore the regulation function of transforming growth factor β（TGF-β1）-induced cardiac fibroblast differentiation by DNA methylation. Methods： Cardiac fibroblasts isolated from neonatal Sprague-Dawley rats were cultured and characterized using immunocytochemistry. First-passage cardiac fibroblasts were used throughout the experiment and stimulated with TGF-β1, DNA methyltransferases（DNMT）, 5-aza-2′-deoxycytidine（5-aza-d C） and TGF-β-neutralizing antibody, respectively. The protein level of α-smooth muscle actin（α-SMA） was determined by Western blot assay and immunofluorescence method. The m RNA levels of α-SMA,DNMT1,DNMT3 a,and DNMT3 b were determined by quantitative polymerase chain reaction,and the global DNMT activity was measured. Results： TGF-β1 and 5-aza-d C both significantly upregulated the expression of α-SMA in cardiac fibroblasts.DNMT1 and DNMT3 a expressions were significantly down-regulated and the global DNMT activity was inhibited when treated with TGF-β1. Conclusion： Cardiac fibroblasts differentiation may be associated with the DNA methylation. Our research provides new insights in cardiac fibrosis from the perspective of epigenetics.

关 键 词：转化生长因子-Β1 心脏成纤维细胞分化 DNA甲基化 DNA甲基转移酶 

分 类 号：R542[医药卫生—心血管疾病]