DNMT3A对高糖环境下小鼠心肌成纤维细胞增殖与迁移的影响

Effects of DNMT3A on mouse cardiac fibroblast proliferation and migration under high glucose environment

目的探讨DNA甲基化转移酶3A(DNA methyltransferases 3A,DNMT3A)在高糖环境下对小鼠心肌成纤维细胞(cardiac fibroblasts,CFs)增殖与迁移的作用。方法取新生C57乳鼠心脏,剪碎后提取CFs,在显微镜下观察并鉴定形态。细胞贴壁后分别在低糖(5.5 mmol·L^(-1))培养基和高糖(33.0 mmol·L^(-1))培养基下培养,并使用DNMT3A慢病毒(沉默DNMT3A基因)处理。应用qRT-PCR方法检测DNMT3A、α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)和Ⅰ型胶原A1(CollagenⅠA1,Col1A1)的mRNA表达变化;应用Western blot方法检测DNMT3A、α-SMA、Col1A1的蛋白表达变化;CCK-8与MTT法检测CFs增殖活性;划痕实验与Transwell迁移实验检测CFs迁移能力。结果与低糖培养的对照组CFs相比,高糖环境诱导了CFs的DNMT3A表达水平升高与胶原纤维沉积,同时CFs的异常活化明显增强。使用慢病毒处理沉默高糖环境下CFs DNMT3A表达后,与空载体组相比,沉默组DNMT3A表达下调,胶原纤维沉积减少,同时CFs的异常增殖与活化明显降低。结论沉默DNMT3A能抑制高糖诱导的胶原沉积与CFs异常活化增殖,DNMT3A通过刺激高糖环境下CFs异常活化与增殖导致糖尿病心肌纤维化和DCM的发生与发展。

Aim To investigate the effect of DNA methyltransferase 3A(DNMT3A)on the proliferation and migration of cardiac fibroblasts(CFs)in C57 mice under high glucose environment.Methods The hearts of C57 mice were taken from 1 to 3 days.After cutting and digesting,CFs were extracted by differential adherance centrifugattion and observed under microscope.After cell attachment,the cells were cultured under low glucose(5.5 mmol·L^(-1))medium and high glucose(33.0 mmol·L^(-1))medium and treated using DNMT3A lentivirus(to silence the DNMT3A gene).qRT-PCR was used to detect mRNA expression of DNMT3A,α-smooth muscle actin(α-SMA)and typeⅠcollagen procollagen A1(Col1A1),the protein expression of DNMT3A,α-SMA and Col1A1 was assayed by Western blot,cell proliferation activity was detected by CCK-8 assay and MTT assay,cell migration ability was detected by scratch assay and Transwell experiment.Results Compared with the low glucose group,the high glucose environment resulted in up-regulated DNMT3A expression in CFs,increased collagen fiber deposition,and promoted CFS proliferation and migration.The expression of DNMT3A was down-regulated in CFs treated with DNMT3A lentivirus under high glucose condition,α-SMA,Col1A1 expression was down regulated,and cell proliferation and migration ability decreased.Conclusions Silencing of DNMT3A could inhibit the proliferation and migration of CFs under high glucose environment,suggesting that DNMT3A may be a key regulatory point in the development of diabetic cardiomyopathy.