目的 观察细胞周期蛋白依赖的蛋白激酶抑制剂4a(INK4a)信号通路对线粒体功能的影响.方法 重组慢病毒载体pLVX-p16INK4a被构建出并感染第3代人骨骼肌成肌细胞,用实时定量反转录聚合酶链反应(RT-qPCR)法与Western blot鉴定p16INK4a基因转录与蛋白表达,7d后,用衰老相关β-半乳糖苷酶染色鉴定细胞衰老程度.同时,用JC-1染料染色和流式细胞仪分别检测线粒体膜电位水平.结果 成功转染了p16INK4a的成肌细胞出现明显衰老表型,其细胞线粒体膜电位为(-40.287±12.663)mV,较阳性对照(CV)及阴性对照(NC)组的(-9.267±1.332)mV和(-6.967±2.031)mV明显下降(P=0.000),而p16INK4a蛋白和mRNA相对表达量分别为45.25±8.21和33.89±7.87,明显高于CV组的10.18±2.69和5.26±1.92及NC组的9.41±1.70和7.16±1.86(P=0.000).结论 INK4a信号通路的上调导致了人骨骼肌成肌细胞的衰老,INK4a通路是人骨骼肌成肌细胞衰老的直接诱因;另外,INK4a通路能对线粒体通路产生作用,使线粒体膜电位下降,激发线粒体通路,间接加速成肌细胞的衰老.
Objective To study the effect of the inhibitor of cyclin-dependent kinase 4a (INK4a) signaling pathway on myoblastic aging.Methods We transfected human skeletal muscle myoblasts with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to identify p16INK4a gene transcription and protein expression.Senescence-associated β-galactosidase staining was used to assess the degree of cell senescence.The mitochondrial membrane potential was analyzed by JC-1 staining and flow cytometry.Results We demonstrated that a senescence phenotype was evident in myoblasts transfected with p16INK4a, the analysis of mitochondrial membrane potential after JC-1 staining by flow cytometry showed a marked decrease in p16 group (-40.287±12.663) mV, while it was (-9.267±1.332) mV in CV group and (-6.967±2.031) mV in NC group.The relative amount of p16 protein and mRNA in p16 group was 45.25±8.21 and 33.89±7.87, which was more than that in CV (10.18±2.69 and 5.26±1.92)and NC (9.41±1.70 and 7.16±1.86) groups (P=0.000).ConclusionThese findings indicated that upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts.Moreover, INK4a signaling pathway activates the mitochondrial pro-aging pathway by reducing the mitochondrial membrane potential, which indirectly accelerates aging in myoblasts.