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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 induce the differentiation of cardiac fibroblasts by transforming growth factor-β1 (TGF-β1) in vitro and to explore the role of DNA methylation. Methods: Isolated and cultured SD rat cardiac fibroblasts were identified by immunocytochemistry. The P1 generation cells were treated with TGF-β1, 5-aza-2’-deoxycytidine (5-aza-d C), a DNA methyltransferase (DNMT) ) And TGF-β neutralizing antibody. The expression of α-smooth muscle actin (α-SMA) was detected by Western blot and immunofluorescence assay. The gene expression of α-SMA and DNMT1, DNMT3a and DNMT3b were detected by real-time quantitative PCR and the DNMT activity was measured. Results: Compared with the control group, TGF-β1 induced a-SMA expression in cardiac fibroblasts as well as 5-aza-d C, and TGF-β1 significantly inhibited total DNMT activity (P <0.05) (P <0.01), but had no significant effect on the expression of DNMT3b (P> 0.05). Conclusion: The differentiation of cardiac fibroblasts may be related to the regulation of DNA methylation modification. This study provides a new therapeutic target for the prevention and treatment of myocardial fibrosis from the perspective of epigenetics.