细胞周期蛋白依赖的蛋白激酶抑制剂4a信号通路对人骨骼肌成肌细胞衰老的诱导及对线粒体膜电位的调控

The inhibitor of cyclin - dependent kinase 4a signaling pathway induces aging in human skeletal muscle myoblasts and decreases the mitochondrial membrane potential

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-p16^INK4a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p16^INK4a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p16^INK4a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p16^INK4a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 myo- blasts with a recombinant lentiviral vector, pLVX -p16INK4a, encoding the p16INx4a gene, and real -time quantitative reverse transcriptase - polymerase chain reaction （ RT - qPCR） and Western blotting were used to identify p16INx4a gene transcription and protein expression. Senescence - associated 13 - galactosidase staining was used to assess the degree of cell senescence. The mitochondrial membrane potential was ana- lyzed 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 and7. 16±1.86） groups （P=0.000）. Conclusion These findings indicated that upregnlation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activates the mitoehondrial pro -aging path- way by reducing the mitochondrial membrane potential, which indirectly accelerates aging in myoblasts.