【摘要】 目的 探讨甲基转移酶(METTL)3调节皮肤成纤维细胞衰老的机制。方法 酶消化法提取小鼠原代皮肤成纤维细胞，对照(Control)组、D-半乳糖(D-gal)组、转染阴性对照siRNA(NC)组、转染METTL3 siRNA(si-METTL3)组、转染核苷二磷酸连接部分X型基元(NUDT)18 siRNA(si-NUDT18)组，D-gal(20 g/L)建立皮肤成纤维细胞衰老模型。采用衰老相关β-半乳糖苷酶(SA-β-gal)染色法检测细胞衰老，实时荧光定量聚合酶链反应(qRT-PCR)检测METTL3 mRNA表达。SA-β-gal染色法、5-乙炔基-2′-脱氧尿苷(EdU)染色法检测si-METTL3组细胞衰老与增殖。蛋白质质谱分析D-gal组与si-METTL3组中的差异蛋白，qRT-PCR验证差异基因成熟与前体的表达水平。SA-β-gal染色法、EdU染色法、JC-1线粒体膜电位检测法和共聚焦显微镜检测线粒体自噬法分别检测si-NUDT18组细胞衰老、增殖、线粒体膜电位和线粒体自噬的表达变化。结果 METTL3 mRNA在衰老皮肤成纤维细胞中表达明显降低，低表达METTL3后细胞衰老水平明显增加，增殖能力明显降低(均P<0.01)。D-gal组与si-METTL3组中NUDT18 mRNA表达明显低于Control组及NC组(P<0.01),pre-NUDT18 mRNA表达无统计学差异(P>0.05)。与NC组相比，si-NUDT18组皮肤成纤维细胞衰老水平明显增加，细胞增殖能力、线粒体膜电位和线粒体自噬水平明显降低(均P<0.01)。结论 METTL3下调可能通过影响NUDT18 mRNA的剪接过程促进皮肤成纤维细胞衰老，抑制增殖及线粒体功能。更多还原

【Abstract】 Objective To study the mechanism of methyltransferase(METTL)3 regulating senescence of skin fibroblasts.Methods Primary mouse skin fibroblasts were extracted by enzyme digestion, and divided into Control group, D-galactose(D-gal) group, negative control siRNA transfection(NC) group, METTL3 siRNA transfection(si-METTL3) group and nucleoside diphosphate linked moiety X motif(NUDT)18 siRNA transfection(si-NUDT18) group. Skin fibroblasts senescence model was established by D-gal(20 g/L), cell senescence was detected by senescence-associated β-galactosidase(SA-β-gal) staining, and METTL3 mRNA expression was detected by quantitative real time polymerase chain reaction(qRT-PCR). SA-β-gal staining and 5-Ethynyl-2’-deoxyuridine(EdU) staining were used to detect cell senescence and proliferation in si-METTL3 group. The differential protein expressions in D-gal and si-METTL3 groups were analyzed by protein spectrum, and qRT-PCR was used to verify the expression levels of differential gene maturation and precursors. SA-β-gal staining, EdU staining, JC-1 mitochondrial membrane potential detection and confocal microscopy detection of mitochondrial autophagy were used to detect the expression changes in cellular senescence, proliferation, mitochondrial membrane potential, and mitophagy of si-NUDT18 group, respectively.Results The expression of METTL3 mRNA was significantly decreased in senescent skin fibroblasts, and the senescence level was significantly increased and proliferation ability was significantly decreased after METTL3 low expression(all P<0.01). NUDT18 mRNA expression was significantly decreased in D-gal group and si-METTL3 group than that in Control group and NC group(P<0.01), while there was no statistical difference in pre-NUDT18 mRNA expression(P>0.05). Compared with NC group, skin fibroblast senescence level was significantly increased, cell proliferation ability, mitochondrial membrane potential and mitochondrial autophagy levels were significantly decreased in si-NUDT18 group(all P<0.01).Conclusions METTL3 down-regulation might promote senescence of skin fibroblasts and inhibit proliferation and mitochondrial function by influencing the splicting process of NUDT18 mRNA.更多还原