关键词: 程序性死亡配体1, 牙髓干细胞, 衰老, 氧化应激, 线粒体膜电位

Abstract:

Objective To investigate the effect of programmed death-ligand 1 (PD-L1) on the aging process of dental pulp stem cells (DPSCs) and its regulatory mechanism, so as to provide a basis for optimizing the clinical application strategy of DPSCs. Methods Primary DPSCs were isolated and cultured by enzyme digestion method. Oxidative stress-induced senescence model was constructed by H₂O₂ induction, and a replicative senescence model was established by continuous subculture. The expression differences of PD-L1 in young and senescent DPSCs were detected by Western blot, RT-qPCR and immunofluorescence. High-efficiency PD-L1 interference sequences were screened to construct PD-L1 knockdown cell models. SA-β-gal staining, RT-qPCR and Western blot were used to detect the senescence phenotype of DPSCs and the expression of senescence-related markers such as P16, P21 and P53. The expression levels of odontogenesis/osteogenesis-related proteins such as DMP1, DSPP, and ALP were detected by Western blot, and further ALP staining was used to examine the effect of PD-L1 knockdown on the ALP activity of DPSCs. ROS detection and JC-10 staining were performed to evaluate the effect of PD-L1 on the oxidative stress level and mitochondrial function of DPSCs. Results Compared with young DPSCs, the mRNA and protein expression levels of PD-L1 in senescent DPSCs were significantly increased (P<0.01). After PD-L1 knockdown, the proportion of SA-β-gal positive cells was significantly reduced (P<0.001); the expression of P16, P21 and P53 was down-regulated (P<0.01); the expression levels of ALP, DMP1 and DSPP were significantly up-regulated (P<0.001); intracellular ROS accumulation was reduced (P<0.001); mitochondrial membrane potential was increased (P<0.001), and mitochondrial function was improved. Conclusion Knockdown of PD-L1 can effectively alleviate the senescence process of DPSCs and significantly promote their odontogenic/osteogenic differentiation potential; its regulatory mechanism may be closely related to reducing intracellular oxidative stress level and improving mitochondrial membrane potential.

Key words: programmed death-ligand 1 (PD-L1), dental pulp stem cells (DPSCs), senescence, oxidative stress, mitochondrial membrane potential