Notch信号介导骨细胞过表达BMP4促进骨髓基质细胞成骨分化  

Notch signaling-mediated osteocytic overexpression of BMP4 promotes osteogenic differentiation of bone marrow stromal cells

作　　者：谢正松 赵怡心 吕子灵 刘宏[2] 涂小林[1] XIE Zhengsong;ZHAO Yixin;LYU Ziling;LIU Hong;TU Xiaolin(Laboratry of Bone Development and Regeneration,Institute of Life Sciences,Chongqing Medical University,Chongqing,400016,China;Center of Reproduction Medicine and Infertility,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China;the First Clinical College of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学生命科学研究院骨发育与再生实验室,重庆400016 [2]重庆医科大学附属第一医院生殖健康与不孕症中心,重庆400016 [3]重庆医科大学第一临床学院,重庆400016

出　　处：《第三军医大学学报》2021年第16期1550-1558,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672118);重庆市科委基础科学与前沿研究技术专项重点课题(cstc2015jcyjBX0119)。

摘　　要：目的探究骨细胞MLO-Y4过表达骨形态发生蛋白4(bone morphogenetic protein-4,BMP4)通过Notch信号对骨髓基质细胞ST2成骨分化的影响。方法使用BMP4重组腺病毒(Ad-BMP4)以及阴性对照(Ad-GFP)分别感染MLO-Y4后与ST2共培养,分为GFP组及BMP4组;Western blot检测MLO-Y4细胞BMP4蛋白表达;采用实时荧光定量PCR(qPCR)检测MLO-Y4的BMP4和Notch配体基因、共培养体系中成骨标志基因、Notch靶基因以及促血管生成因子表达;碱性磷酸酶(alkaline phosphatase,ALP)染色及生化定量检测ALP活性;在共培养体系中加入Notch信号抑制剂DAPT后,再次检测上述指标。结果MLO-Y4过表达BMP4,上调Notch配体Jag1、Jag2、Dll4表达;ALP染色以及生化定量结果显示共培养第1天GFP组和BMP4组ALP活性差异无统计学意义,在第3、5天,BMP4组ALP活性更高(P<0.05);BMP4组成骨标志基因在第1天仅Runt相关转录因子2(runt-related transcription factor 2,Runx2)显著升高(P<0.05),第3、5天BMP4组Alp、Runx2、骨钙蛋白(osteocalcin,Ocn)、Ⅰ型胶原(collagenⅠ)均显著增高(P<0.05);第3天BMP4组Notch信号靶基因Hey1、Hey2,血管内皮生长因子(vascular endothlial growth factor,VEGF)、低氧诱导因子(hypoxia-inducible factor 1α,HIF1α)的表达也显著增高(P<0.05);加入DPAT抑制Notch信号后,BMP4组ALP活性、成骨标志基因以及促血管生成因子表达均显著下降(P<0.05)。结论MLO-Y4过表达BMP4可以促进ST2成骨分化,该过程可能与Notch信号增强有关。Objective To investigate the effect of bone morphogenetic protein 4(BMP4)overexpression in MLO-Y4 osteocytes on osteogenic differentiation of bone marrow stromal ST2 cells through Notch signaling.Methods Ad-GFP or Ad-BMP4 infected MLO-Y4 cells were co-cultured with ST2 cells,and designated as GFP and BMP4 group respectively.Western blotting was used to detect the BMP4 protein level in MLO-Y4 cells.Real-time fluorescence quantitative analysis(qPCR)was applied to determine the mRNA expression of BMP4 and Notch ligands genes as well as osteoblast genes in the MLO-Y4 cells,and Notch target genes and angiogenesis factors in the co-cultured systems.The activity of alkaline phosphatase(ALP)was determined by ALP staining and biochemical quantification.Notch signal inhibitor DAPT was added to the co-culture system to detect the above indexes again.Results Overexpression of BMP4 upregulated the expression of Notch ligands Jag1,Jag2 and Dll4 in MLO-Y4 cells.ALP staining and biochemical quantitative results demonstrated that there was no significant difference in ALP activity at day 1 between the GFP and BMP4 groups,and stronger ALP activity was seen in the latter group at day 3 and 5(P<0.05).In the BMP4 group,at day 1 among the osteoblast genes,only runt-related transcription factor 2(Runx2)was obviously expressed(P<0.05),and the levels of Alp,Runx2,osteocalcin(Ocn),and collagenⅠwere all highly expressed in the BMP4 group(P<0.05).At day 3,Notch signaling target genes Hey1 and Hey2,angiogenesis factors vascular endothelial growth factor(VEGF)and hypoxia-inducible factor 1(HIF1α)were also highly expressed in the BMP4 group compared with the control group(P<0.05).After addition of DAPT to inhibit Notch signaling,the activity of ALP and expression of osteoblast genes and angiogenesis genes were all decreased(P<0.05).Conclusion Overexpression of BMP4 in MLO-Y4 cells promotes osteogenic differentiation of ST2 cells,which may be related to the enhancement of Notch signaling.

关 键 词：骨细胞 骨形态发生蛋白4 骨髓基质细胞 成骨分化 NOTCH信号 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]