缺氧诱导因子1α调控wnt/β-catenin信号通路对常氧条件下大鼠髓核细胞衰老的作用及机制  

Effect and mechanism of HIF-1α regulating wnt/β-catenin signaling pathway on senescence of nucleus pulposus cells in rats under normal oxygen

作　　者：雷淼 邹略滔 蔡伟业 董伟 江维[1] 胡侦明[1] LEI Miao;ZOU Lüetao;CAI Weiye(Department of Orthopedics,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400010,China)

机构地区：[1]重庆医科大学附属第一医院骨科,重庆市400010 [2]重庆医科大学附属第三医院骨科,重庆市400010 [3]西南医科大学,四川省泸州市646000

出　　处：《中国脊柱脊髓杂志》2023年第9期815-822,共8页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金(82002363);重庆市自然科学基金(cstc2020jcyj-msxmX0195)。

摘　　要：目的:探究缺氧诱导因子1α(HIF-1α)调控wnt/β-catenin信号通路对常氧培养下大鼠髓核细胞衰老的影响及作用机制.方法:取5只4周龄雌性SD大鼠,提取鼠尾髓核原代细胞进行研究.(1)将髓核细胞分为5组:转染对照组[用磷酸盐缓冲盐水(PBS)处理髓核细胞]、空载腺病毒组(用空载腺病毒处理髓核细胞)、过表达HIF-1α组(用载过表达HIF-1α质粒的腺病毒处理髓核细胞)、空载siRNA组(用空载siRNA处理髓核细胞)、敲减HIF-1α组(用siRNA敲减HIF-1α处理髓核细胞),在常氧下培养48h后,免疫蛋白印迹(western blot,WB)检测HIF-1α和衰老相关基因p53、p21、p16,β-gal染色检测细胞衰老,评估常氧条件下HIF-1α对于髓核细胞衰老的影响.(2)取髓核细胞,分为空载腺病毒组、过表达HIF-1α组、空载siRNA组、敲减HIF-1α组,处理方式同前,在常氧下培养48h后,WB检测HIF-1α、GSK-3β和β-catenin通路的表达.取髓核细胞,分为对照组(用PBS处理髓核细胞)、过表达HIF-1α组(用载过表达HIF-1α质粒的腺病毒处理髓核细胞)、XAV-939组(用XAV-939处理髓核细胞)、XAV-939+过表达HIF-1α组(用XAV-939+载过表达HIF-1α质粒的腺病毒处理髓核细胞),WB检测HIF-1α、p53、p21、p16和wnt/β-catenin的表达,β-gal染色检测细胞衰老,以检测HIF-1α与wnt/β-catenin信号通路的关系以及对髓核细胞衰老的影响.结果:(1)腺病毒转染HIF-1α后,与转染对照组和空载腺病毒组相比,过表达HIF-1α组HIF-1α表达增加(P<0.05).小干扰RNA转染HIF-1α后,与转染对照组和空载siRNA组相比敲减HIF-1α组HIF-1α表达降低(P<0.05).WB结果显示,与空载腺病毒组相比过表达HIF-1α组HIF-1α表达增加(P<0.05),而p53、p21和p16表达降低(P<0.05),与空载siRNA组相比敲减HIF-1α组HIF-1α表达降低(P<0.05),而p53和p16表达增加(P<0.05).β-gal染色显示,在常氧条件下培养48h,与空载腺病毒组相比,过表达HIF-1α组衰老细胞降低(P<0.05),与空载siRNA组相比Objectives:To investigate the effect and mechanism of hypoxia-inducing factor 1α(HIF-1α)regulating wnt/β-catenin signaling pathway on senescence of nucleus pulposus cells in rats under normal oxygen.Methods:The primary cells of caudal nucleus pulposus were extracted from 5 female SD rats of 4 weeks old.(1)The nucleus pulposus cells were divided into the following 5 groups:transfection control group[treated with phosphate buffered saline(PBS)],adenovirus unloaded group(treated with unloaded adenovirus),and HIF1α over-expression group(treated with adenovirus carrying over-expressing HIF-1α plasmid),unloaded siRNA group(treated with unloaded siRNA),and HIF-1α knockdown group(treated with siRNA knocked HIF-1α).HIF-1α,p53,p21,and p16 were detected by Western blot(WB)after normal oxygen culture for 48h,and cell senescence was detected by β-gal staining to evaluate the effect of HIF-1α on nucleus pulposus cell senescence under normal oxygen culture.(2)Nucleus pulposus cells were extracted and divided into adenovirus unloaded group,HIF-1α over-expression group,unloaded siRNA group,and HIF-1αknockdown group,with same treatment as mentioned above for each group.And HIF-1α,GSK-3β,and β-catenin pathway expressions were detected by WB test.Nucleus pulposus cells were extracted and divided into control group(treated with PBS),HIF-1α over-expression group(treated with adenovirus carrying over-expressing HIF-1α plasmid),XAV-939 group(treated with XAV-939),XAV-939+HIF-1α over-expression group(treated with XAV-939+adenovirus carrying over-expressing HIF-1α plasmid).WB test was used to detect HIF-1α,p53,p21,p16,and wnt/β-catenin signal expressions and β-gal staining was applied to detect cell senescence to investigate the relationship between HIF-1α and wnt/β-catenin signalpathway and the effect of HIF-1α on senescence of nucleus pulposus cells.Results:(1)After adenovirus transfection of HIF-1α,HIF-1α expression increased in HIF-1α over-expression group comparing with transfection control group and adeno

关 键 词：缺氧诱导因子1Α WNT/Β-CATENIN信号通路 氧气浓度 髓核细胞衰老 

分 类 号：Q593[生物学—生物化学] R681.5[医药卫生—骨科学]