雷 淼,邹略滔,蔡伟业,董 伟,江 维,胡侦明.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[J].中国脊柱脊髓杂志,2023,(9):815-822.
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
中文关键词:  缺氧诱导因子1α  wnt/β-catenin信号通路  氧气浓度  髓核细胞衰老
中文摘要:
  【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,敲减HIF-1α组衰老细胞数量则增加(P<0.001)。(2)与空载腺病毒组相比,过表达HIF-1α组β-catenin表达升高(P<0.01),同时GSK-3β表达降低(P<0.05)。与空载siRNA组相比敲减HIF-1α组β-catenin表达降低(P<0.0001),同时GSK-3β表达升高(P<0.05)。与对照组相比,XAV-939组β-catenin表达降低(P<0.001),p53、p21和p16的表达升高,XAV-939+过表达HIF-1α组β-catenin表达降低(P<0.05),p21表达升高(P<0.05)。与过表达HIF-1α组相比XAV-939+过表达HIF-1α组衰老细胞数增加(P<0.001),与对照组相比过表达HIF-1α组的细胞核内β-catenin表达更多,而XAV-939组和XAV-939+过表达HIF-1α组则相对较少(P<0.05)。结论:HIF-1α通过激活wnt/β-catenin信号通路抑制常氧条件培养下大鼠髓核细胞的衰老。
Effect and mechanism of HIF-1α regulating wnt/β-catenin signaling pathway on senescence of nucleus pulposus cells in rats under normal oxygen
英文关键词:Hypoxia-inducing factor 1α  wnt/β-catenin signaling pathway  Oxygen concentration  Nucleus pulposus cell senescence
英文摘要:
  【Abstract】 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 HIF-1α 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-1α knockdown 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 adenovirus unloaded group(P<0.05). After siRNA transfection of HIF-1α, HIF-1α expression decreased in HIF-1α knockdown group than transfection control group and siRNA unloaded group(P<0.05). WB result showed that comparing with adenovirus unloaded group, HIF-1α expression increased in HIF-1α over-expression group(P<0.05), while the expressions of p53, p21, and p16 decreased(P<0.05); Comparing with siRNA unloaded group, H1F-1α expression decreased in HIF-1α knockdown group(P<0.05), while the expressions of p53 and p16 increased(P<0.05). β-gal staining showed culture 48h under normal oxygen condition, the number of senescence cells decreased in HIF-1α over-expression group(P<0.05) than adenovirus unloaded group and increased than siRNA unloaded group(P<0.001). (2)In comparison with adenovirus unloadedgroup, HIF-1α over-expression group increased in β-catenin expression(P<0.01) and decreased in GSK-3β expression(P<0.05); In comparison with siRNA unloaded group, HIF-1α knockdown group decreased in β-catenin expression(P<0.0001) and increased in GSK-3β expression(P<0.05). Comparing with the control group, β-catenin expression in XAV-939 group decreased(P<0.001), and the expressions of p53, p21, and p16 increased; β-catenin expression in XAV-939+ HIF-1α over-expression group decreased(P<0.05) and p21 expression increased(P<0.05). The number of senescence cells in XAV-939+HIF-1α over-expression group was bigger than that in HIF-1α over-expression group(P<0.001), and β-catenin expression in HIF-1α over-expression group was more than the control group, while that in XAV-939 group and XAV-939+HIF-1α over-expression group was less(P<0.05). Conclusions: HIF-1α inhibits senescence of rat nucleus pulposus cells cultured under normal oxygen by activating wnt/β-catenin signaling pathway.
投稿时间:2023-01-16  修订日期:2023-08-10
DOI:
基金项目:国家自然科学基金(82002363);重庆市自然科学基金(cstc2020jcyj-msxmX0195)
作者单位
雷 淼 重庆医科大学附属第一医院骨科 400010 重庆市 
邹略滔 重庆医科大学附属第三医院骨科 400010 重庆市 
蔡伟业 西南医科大学 646000 四川省泸州市 
董 伟  
江 维  
胡侦明  
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