ALKBH5调控大鼠心肌成纤维细胞活化增殖的作用研究

Effects of ALKBH5 regulation on proliferation of cardiac fibroblasts in rats

目的探讨6-甲基腺嘌呤(m^(6)A)去甲基化酶ALKBH5在大鼠心肌成纤维细胞(CFs)活化增殖中的作用。方法取1~3 d新生SD乳鼠心脏,剪碎消化后进行CFs原代培养,并在显微镜下观察细胞形态。细胞贴壁生长后加入TGF-β1诱导构建CFs活化增殖模型,模型构建成功后分别向各组细胞转染ALKBH5(慢病毒过表达ALKBH5)及慢病毒空载体24~48 h。运用RT-qPCR方法检测ALKBH5、α-平滑肌肌动蛋白(α-SMA)、I型胶原(CollagenⅠ)和增殖细胞核抗原(PCNA)mRNA的表达;Western blot检测ALKBH5、α-SMA、CollagenⅠ及PCNA蛋白的表达;CCK-8法和EdU染色检测细胞增殖活性变化。结果在CFs活化增殖模型中,与对照组CFs相比,模型组CFs中ALKBH5蛋白和mRNA的表达降低,而与活化增殖相关蛋白PCNA、α-SMA及CollagenⅠ的表达增加。此外,在慢病毒转染ALKBH5过表达组的CFs中,α-SMA、CollagenⅠ和PCNA蛋白和mRNA同ALKBH5病毒空载体组相比表达下降。CCK-8与EdU染色实验表明,与空载体组相比,ALKBH5过表达组CFs增殖活性受到抑制。结论过表达ALKBH5明显抑制CFs的增殖活性,提示ALKBH5可能是参与调控CFs活化增殖的关键因子。

Objective To investigate the role of N-methyladenosine(m^(6)A)demethylase ALKBH5 in the proliferation and activation of cardiac fibroblasts(CFs)in rats.Methods The CFs taken from SD rats in 1 to 3 days were isolated by differential adhesion and observed under microscope.After cells were adherently grown to appropriate density,the cells were induced by TGF-β1 for modeling.The model cells were divided into the overexpression of ALKBH5 group infected by lentivirus and the negative control group for 24-48 hours.RT-qPCR was used to detect mRNA expression of ALKBH5,α-smooth muscle actin(α-SMA),type I collagen(CollagenⅠ)and proliferating cell nuclear antigen(PCNA).The expression of ALKBH5、α-SMA、CollagenⅠand PCNA were assayed by Western blot.The cell proliferation activity was tested by CCK-8 assay and EdU.Results Compared with the control group,the protein and mRNA of ALKBH5 were reduced in the model group active by TGF-β1.Meanwhile,the biomarkers of activation,such as PCNA,α-SMA and CollagenⅠ,increased significantly.Besides,the protein and mRNA of PCNA、α-SMA and CollagenⅠwere lower in overexpression of ALKBH5 group than those of the negative control group.CCK-8 assay and EdU suggested that the proliferation viability of CFs was reduced evidently in overexpression of ALKBH5 group,compared with the negative control group.Conclusion Overexpression of ALKBH5 can inhibit the proliferation of CFs,suggesting that ALKBH5 may be a key regulatory point in the development of myocardial fibrosis.