摘要
目的目前关于甲基化对心肌纤维化形成的作用机制仍不明确。文章旨在探讨DNA甲基化转移酶3A(DNMT3A)对心肌成纤维细胞活化过程中胶原表达变化的调控。方法选取50只乳鼠,提取培养SD乳鼠心肌成纤维细胞(CFs),进行细胞转染,根据CFs转染的试剂分为5组:DNMT3A组(CFs转染重组的DNMT3A基因过表达质粒)、空载体组(CFs转染p EGFP-C3空载体质粒)、DNMT3A siRNA组(CFs转染小干扰DNMT3A siRNA)、DNMT3A siRNA NC组(CFs转染DNMT3A siRNA NC空载体质粒)、空白对照组(CFs不作转染处理)。ELISA检测空白对照组、DNMT3A组、DNMT3A siRNA组细胞上清液胶原变化。qRT-PCR检测5组相关mRNA的表达,Western blot法检测除DNMT3A siRNA NC组外其余4组CFs中ColⅠ、ColⅢ和DNMT3a蛋白的表达及CCK8法检测4组细胞的增殖活性。结果 DNMT3A组的Ⅰ型胶原和Ⅲ型胶原明显高于空白对照组(P<0.05),DNMT3A siRNA组的Ⅰ型胶原和Ⅲ型胶原的含量明显低于空白对照组(P<0.05)。DNMT3A组的ColⅠ、ColⅢ及DNMT3A表达量较空白对照组、空载体组明显升高(P<0.05),而DNMT3A siRNA组明显低于空白对照组及空载体组(P<0.05)。DNMT3A组细胞活力(2.087±0.317)明显高于空载体组(1.063±0.223)、空白对照组(1.082±0.207),差异有统计学意义(P<0.05); DNMT3A siRNA组(0.463±0.087)明显低于空载体组、空白对照组(P<0.05)。结论 DNMT3A可以促进CFs的活化增殖,并且能够增加胶原的表达,从而对心肌纤维化产生促进作用。
Objective The mechanisms of methylation acting on myocardial fibrosis are not yet clear at present.The aim of this study was to investigate the role of DNA methyltransferase 3A(DNMT3A)in regulating the expressions of collagens during the activation of cardiac fibroblasts. Methods Cardiac fibroblasts were obtained from 50 neonatal mice and divided into three groups:blank control,DNMT3A overexpression plasmid(mDNMT3A-pEGFP-C3)and small interference DNMT3A siRNA.The contents of collagens in the cell supernatant were detected by ELISA.The mRNA and protein expressions of type I collagen(ColⅠ),typeⅢcollagen(ColⅢ)and DNMT3A in the cardiac fibroblasts were determined by real-time quantitative PCR and Western blot respectively and the proliferative activity of the cardiac fibroblasts measured by CCK8 assay. Results The contents of Col I and ColⅢin the cell supernatant were significantly increased in the DNMT3A overexpression plasmid group but decreased in the DNMT3A siRNA group as compared with those in the blank control(P<0.05).The expressions of ColⅠ,ColⅢand DNMT3A were remarkably higher in the DNMT3A overexpression plasmid group but lower in the DNMT3A siRNA group than in the blank control(P<0.05).The cell activity was markedly higher in the DNMT3A overexpression plasmid group than in the empty vector plasmid and control groups(2.087±0.317 vs 1.063±0.223 and 1.082±0.207,P<0.05)but lower in the DNMT3A siRNA group(0.463±0.087)than in the latter two(P<0.05). Conclusion DNMT3A can increase the proliferation and activation of cardiac fibroblasts,upregulate the expressions of collagens and thus promote myocardial fibrosis.
作者
代晨
陶辉
石开虎
徐盛松
DAI Chen;TAO Hui;SHI Kai-hu;XU Sheng-song(Department of Cardiothoracic Surgery,the Second Affiliated Hospital of Anhui Medical University,Cardiovascular Research Center,Anhui Medical University,Hefei 230601,Anhui,China)
出处
《医学研究生学报》
CAS
北大核心
2018年第12期1237-1241,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81570295)
