复制性老化髓核细胞Hippo-Yes相关蛋白信号通路的作用机制

摘要

Objective To investigate the mechanism of Hippo-Yes-associated protein (YAP)signaling pathway in replicative senescent nucleus pulposus cells (NPCs).Methods The human nucleus pulposus cells (NPCs) as the research subject,and the groups were divided by the aging characteristics and different interfering lentivirus,A group;the third generation NPCs with non-senecence,B group;senescent NPCs,and subdivide different subgroups according to the transfection of different lentiviral vectors (shYAP and sh-Control).The third generation of NPCs were cultured in vitro and induced replication senescence (RS) cells model through continuous passage,senescent cell was performed by SA-β-Gal staining method and the NPCs biological characteristics and senescent rates were compared and analyzed among different subgroups.Results The mean positive percentage of SA-β-Gal staining was significantly elevated in the B group (90.4％) compared with the A group (15.6％) (P＜0.05).A group showed that the expression level of YAP gene and protein was higher than that of B group (0.77 ±0.12 vs.0.31 ±0.04,0.52±0.06 vs.0.12 ±0.05,P＜0.05),and immunofluorescence showed that YAP was mainly located in the nucleus (85％).On the contrary,Hippo-YAP signaling pathway was activated in group B,YAP and its target gene CYR61 decreased and the distribution of YAP was transferred from nucleus to cytoplasm,and the proportion of cytoplasmic YAP increased (56％).In B shYAP and sh-Control groups,they displayed a unique senescent appearance and no obvious effect on the cell senescence (P ＞ 0.05).Conclusion Hippo-YAP signaling pathway is involved in the development of NPCs senescence and plays an important role in maintaining cell growth and proliferation.%目的 探讨Hippo-Yes相关蛋白(YAP)信号通路在复制性老化髓核细胞中的作用机制.方法 取人源髓核细胞(NPCs)为研究对象,根据老化特性及慢病毒干扰载体不同进行分组,即A组:未老化第三代NPCs,B组:第十代老化NPCs,A和B两组根据转染不同的慢病毒载体shYAP或sh-Control再分两个亚组,分别是A sh-Control组,A shYAP组,B sh-Control组和B shYAP组.体外培养第三代NPCs,通过连续传代诱导建立复制性老化细胞模型(RS),细胞衰老SA-β-Gal染色检测分析老化与未老化NPCs生物学特性.慢病毒载体shYAP和sh-Control分别转染老化和未老化NPCs,检测Hippo-YAP信号通路干预情况及对下游靶基因CYR61的表达影响.最后,SA-β-Gal染色检测分析各个亚组老化与未老化NPCs生物学特性和老化率.结果 B组SA-β-Gal染色显示NPCs蓝色团块数目高于A组(90.4％比15.6％,P＜0.05);A组可见YAP基因和蛋白水平表达高于B组(0.77 ±0.12比0.31±0.04,0.52±0.06比0.12±0.05,P＜0.05),免疫荧光显示YAP主要位于细胞核内(85％).B组细胞Hippo-YAP信号通路激活,YAP及其靶基因CYR61的基因水平和蛋白水平降低,且YAP分布由核内转移至胞质内,胞质YAP比例增加(56％).B组中两亚组细胞均显示出老化细胞特有的的外观表现,shYAP慢病毒载体转染老化NPCs后对其细胞老化率无明显影响(P＞0.05).结论 Hippo-YAP信号通路参与NPCs老化的发生发展,对维持细胞的生长、增殖发挥着重要作用.