IKKβ缺失和NF-κB p65缺失对小鼠成骨细胞分化的影响  

Effects of IKKβ or NF-κB p65 deficiency on differentiation of mouse osteoblasts

作　　者：郝倩云 刘壮壮 周斌辉 冯心雨 黄蓉 HAO Qianyun;LIU Zhuangzhuang;ZHOU Binhui;FENG Xinyu;HUANG Rong(Department of Thoracic OncologyⅡ,Key Laboratory of Carcinogenesis and Translational Research(Ministry of Education/Beijing),Peking University Cancer Hospital&Institute,Beijing 100142;Institute of Psychiatry and Neuroscience,Xinxiang Medical University,Xinxiang,Henan 453000;School of Laboratory Medicine,Xinxiang Medical University,Xinxiang,Henan 453000)

机构地区：[1]北京大学肿瘤医院暨北京市肿瘤防治研究所胸部肿瘤内二科,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142 [2]新乡医学院精神神经医学研究院,河南新乡453000 [3]新乡医学院医学检验学院,河南新乡453000

出　　处：《郑州大学学报（医学版）》2022年第3期324-329,共6页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金青年科学基金项目(81901581)。

摘　　要：目的:探讨IKKβ和NF-κB p65缺失对小鼠成骨细胞分化的影响。方法:使用CRISPR/Cas9基因编辑工具分别敲除小鼠前成骨细胞MC3T3-E1中的Ikbkb或Rela基因,获得不表达IKKβ或NF-κB p65蛋白的Ikbkb^(-/-)MC3T3-E1和Rela^(-/-)MC3T3-E1细胞。用10 mmol/Lβ-甘油磷酸+50 mg/L抗坏血酸诱导MC3T3-E1、Ikbkb^(-/-)MC3T3-E1、Rela^(-/-)MC3T3-E1细胞成骨分化。诱导培养4 d后,qPCR法检测细胞中成骨分化标志性基因Sp7、Alpl、Spp1、Bglap mRNA的表达,Western blot法检测细胞中成骨分化相关蛋白ALPL、FZD9的表达;16 d后,茜素红染色法检测细胞外基质矿化水平。结果:诱导培养后Ikbkb^(-/-)MC3T3-E1细胞中Sp7、Alpl、Bglap mRNA表达以及ALPL、FZD9蛋白表达高于MC3T3-E1细胞和Rela^(-/-)MC3T3-E1细胞(P<0.010);Ikbkb^(-/-)MC3T3-E1细胞外基质矿化水平高于MC3T3-E1和Rela^(-/-)MC3T3-E1细胞(P<0.010),而Rela^(-/-)MC3T3-E1与MC3T3-E1细胞比较差异无统计学意义(P>0.05)。结论:IKKβ缺失与NF-κB p65缺失在成骨细胞分化过程中的功能可能不同,IKKβ缺失促进成骨细胞分化,而NF-κB p65缺失则无此作用。Aim:To investigate the effects of IKKβor NF-κB p65 deficiency on mouse osteoblast differentiation.Methods:Ikbkb or Rela gene of mouse pre-osteoblast MC3T3-E1 cells were knocked out by CRISPR/Cas9 method to obtain Ikbkb^(-/-)MC3T3-E1 cells(without IKKβprotein expression)and Rela^(-/-)MC3T3-E1 cells(without NF-κB p65 protein expression).MC3T3-E1,Ikbkb^(-/-)MC3T3-E1 and Rela^(-/-)MC3T3-E1 were induced osteogenic differentiation by 10 mmol/Lβ-glycerophosphate+50 mg/L ascorbic acid.After 4 days osteogenic induction,the mRNA of osteoblast differentiation makers including Sp7,Alpl,Spp1,and Bglap were detected by qPCR;the expressions of ALPL and FZD9 protein were detected by Western blot.After 16 days induction,extracellular matrix mineralization was assessed by alizarin red staining.Results:After the induction,the expressions of Sp7,Alpl and Bglap mRNA,and ALPL,FZD9 protein in Ikbkb^(-/-)MC3T3-E1 cells were higher than those in MC3T3-E1 and Rela^(-/-)MC3T3-E1 cells(P<0.010);the mineralization level of Ikbkb^(-/-)MC3T3-E1 cells was higher than those of MC3T3-E1 and Rela^(-/-)MC3T3-E1 cells(P<0.010),but there was no significant difference between Rela^(-/-)MC3T3-E1 and MC3T3-E1 cells(P>0.05).Conclusion:IKKβ-deficiency and NF-κB p65-deficiency have different functions during osteoblast differentiation,IKKβ-deficiency may promote osteoblast differentiation,while NF-κB p65-deficiency has no such function.

关 键 词：成骨细胞 IKKΒ NF-κB p65 NF-κB信号通路 细胞分化调控 小鼠 

分 类 号：R683[医药卫生—骨科学]