核因子-κB配体的受体在Wnt/β-连环蛋白通路促成骨样细胞转化中的作用及意义  

Implication of nuclear factor kappa-B ligand-receptor in promoting osteoid cell transformation in Wingless-related integration site/β-catenin pathway

作　　者：杨明 聂斌 范文娟 钟秋英 李寒 王博[2] 张超 管思明[4] 刘婕 李莎莎 Yang Ming;Nie Bin;Fan Wenjuan;Zhong Qiuying;Li Han;Wang Bo;Zhang Chao;Guan Shiming;Liu Jie;Li Shasha(Department of Geriatrics,the Sixth Hospital of Wuhan,Jianghan University,Wuhan 430015,China;Department of Hepatobiliary-Pancreatic Surgery,the Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China;Department of Geriatric Surgery,the Sixth Hospital of Wuhan,Jianghan University,Wuhan 430015,China;Department of Geriatrics,the Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China)

机构地区：[1]江汉大学附属医院,武汉市第六医院老年病科,430015 [2]华中科技大学同济医学院附属协和医院肝胆胰外科,武汉430022 [3]江汉大学附属医院,武汉市第六医院老年外科,430015 [4]华中科技大学同济医学院附属协和医院老年病科,武汉430022

出　　处：《中华实验外科杂志》2021年第7期1202-1205,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省教育厅科研计划项目(B2019236、B2020230)。

摘　　要：目的:探讨核因子(NF-κB)配体的受体在Wnt/β-连环蛋白(β-catenin)通路促成骨样细胞转化中的作用及其意义。方法:分离健康雄性远交群(SD)大鼠主动脉平滑肌细胞(SMC)并诱导为成骨样细胞,分别用LiCl、LiCl+OPG(0.1 ng/ml)、LiCl+OPG(1.0 ng/ml)、LiCl+OPG(10.0 ng/ml)、LiCl+OPG(100.0 ng/ml)处理成骨样细胞,对照组未行LiCl及OPG处理,LiCl浓度均为20 mmol/L。RT-qPCR检测各组细胞OPG、RANKL转录水平;Von Kossa染色,钙离子含量测定,碱性磷酸酶(ALP)活性测定,骨钙素蛋白质印迹法(Western blot)检测各组细胞钙化程度;免疫组织化学和Western blot检测Wnt/β-catenin通路激活情况。采用单因素方差分析,组内进一步两两比较采用LSD-t检验,两两比较采用t检验。结果:LiCl组OPG的表达(4.35±0.88)稍高于成骨样细胞组(4.12±0.82,t=0.603,P>0.05),RANKL表达则LiCl组(2.61±0.42)显著高于成骨样细胞组(1.52±0.35,t=2.317,P<0.05);OPG/RANKL的比值LiCl(1.67±0.41)组显著低于成骨样细胞组(2.71±0.48,t=2.542,P<0.05)。成骨样细胞的转化实验中,成骨样细胞组及加LiCl+OPG的4组钙离子含量、ALP活性、骨钙素的水平(25.2±5.8、68.5±13.3、61.1±12.8、57.6±11.3、56.3±11.4)、(80.4±10.2、141.2±17.6、125.7±15.3、123.5±15.9、122.7±15.1)、(0.61±0.10、0.82±0.14、0.79±0.13、0.72±0.12、0.73±0.13)均低于LiCl组(99.5±16.5)、(186.0±39.3)、(0.98±0.15),(t=5.971、2.795、2.810、2.829、2.831,P<0.05)、(t=5.623、2.597、2.765、2.791、2.793,P<0.05)(t=4.931、2.379、2.384、2.391、2.392,P<0.05);加LiCl+OPG的4组中钙离子含量、ALP活性、骨钙素的水平差异无统计学意义(P>0.05),均高于成骨样细胞组水平(t=5.362、2.769、2.635、2.633,P<0.05)、(t=5.105、2.758、2.732、2.733,P<0.05)、(t=2.762、2.569、2.566、2.563,P<0.05)。Wnt/β-catenin通路的激活程度检测中,β-catenin的表达水平β-catenin表达水平LiCl组(1.75±0.26)高于成骨样细胞组及加LiCl+OPG的4组(1.32±0.18、1.25±0.17、Objective To investigate the role and significance of nuclear factor-kappa B(NF-κB)ligand receptor in the transformation of osteoid cells through the Wingless-related integration site(Wnt)/β-catenin pathway.Methods Aortic smooth muscle cells(SMC)were isolated from SD rats and induced into osteoblast like cells.Osteoblast like cells were treated with LiCl,LiCl+bone protection element(OPG,0.1 ng/ml),LiCl+OPG(1.0 ng/ml),LiCl+OPG(10.0 ng/ml)and LiCl+OPG(100.0 ng/ml)respectively.The control group was not treated with LiCl or OPG,and the concentration of LiCl was 20 mmol/L.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the transcription levels of OPG and RANKL.The von Kossa staining,calcium content,alkaline phosphatase(ALP)activity,osteocalcin and Western blotting were used to detect the degree of calcification.Immunohistochemistry and Western blotting were used to detect the activation of Wnt/β-catenin pathway.One-way ANOVA was used to compare the differences between the experimental groups and control group,LSD-t test was used for multiple comparison.Results The expression of OPG in LiCl group(4.35±0.88)was slightly higher than that in osteoblast like cell group(4.12±0.82,t=0.603,P>0.05).The expression of RANKL in LiCl group(2.61±0.42)was significantly higher than that in osteoblast like cell group(1.52±0.35,t=2.317,P<0.05).The ratio of OPG/RANKL in LiCl group(1.67±0.41)was significantly lower than that in osteoblast like cell group(2.71±0.48,t=2.542,P<0.05).In the transformation experiment of osteoblast like cells,the calcium content,ALP activity and osteocalcin level in osteoblast like cells group and the LiCl+OPG groups[(25.2±5.8,68.5±13.3,61.1±12.8,57.6±11.3,56.3±11.4),(80.4±10.2,141.2±17.6,125.7±15.3,123.5±15.9,122.7±15.1),(0.61±0.10,0.82±0.14,0.79±0.13,0.72±0.12,0.73±0.13)]were significantly reduced as compared with those in the LiCl group[(99.5±16.5),(186.0±39.3),(0.98±0.15),(t=5.971,2.795,2.810,2.829,2.831,P<0.05),(t=5.623,2.597,2.7

关 键 词：动脉钙化 WNT/Β-CATENIN通路 成骨样细胞 骨保护素 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]