Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨  

Role of Notch Signaling Pathway in Bone Morphogenetic Protein 4-induced Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells and Its Mechanism

作　　者：曹俊杰[1] 李爱芳[1] 卫亚琳 廉静[1] 唐敏[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物工程杂志》2017年第4期48-55,共8页China Biotechnology

基　　金：重庆市自然科学基金(CSTC2012jj A10004;KJ130305)资助项目

摘　　要：目的:探讨Notch信号对骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导间充质干细胞成骨分化的影响以及作用机制。方法:(1)DAPT或Ad-dominant-negative mutants of Notch1(Addn Notch1)和BMP4-CM处理小鼠胚胎成纤维细胞,检测早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP);(2)茜素红S染色实验检测晚期成骨钙盐沉积情况;(3)半定量反转录聚合酶链反应(RT-PCR)检测成骨分化相关基因ALP,Runx2,Col1a1的表达;(4)免疫细胞化学检测p-Smad1/5/8的表达;(5)结晶紫染色和流式细胞术检测细胞的增殖及周期改变。结果:(1)DAPT抑制BMP4诱导的早期成骨分化,且呈浓度依赖性;(2)Delta-like 1(DLL1)促进BMP4诱导的成骨分化,DAPT和dn Notch1抑制BMP4诱导的成骨分化;(3)DLL1促进BMP4诱导的成骨相关基因ALP,Runx2,Col1a1的表达,DAPT抑制这些基因的表达;(4)DLL1促进BMP4诱导的细胞核内p-Smad1/5/8的表达,而DAPT抑制其表达;(5)DLL1促进BMP4诱导的细胞增殖,而DAPT抑制BMP4诱导的细胞增殖。结论:Notch信号通过BMP/Smads信号通路促进BMP4诱导的MSCs成骨分化,在此过程中也有促细胞增殖的作用。Objective： To study the role of Notch signaling pathway in bone morphogenetic protein 4 （BMP4） induced osteogenic differentiation of mesenchymal stem cells and its mechanism. Method ： （ 1 ） After treatment MEFs cells with DAPT or AddnNotchl and BMP4-CM, the early osteogenic marker alkaline phosphatase （ALP） activity was detected by quantitative assay and staining assay; （2） Later osteogenic marker calcium deposition was determined by Alizarin Red S staining; （3） The expression of essential osteogenic factors （ALP, Runx2, Collal ） induced by BMP4 were detected by RT-PCR; （4） The protein expression of p-Smadl/ 5/8 was detected by Immunohistochemistry （ICC） ; （5） The cell proliferation, cycle were determined with crystal violet staining and flow cytometry. Results： （1） BMP4-induced early osteogenic marker ALP activity was significantly inhibited by DAPT in a dose-dependent manner; （2） After transfecting AdDLL1 and treating MEFs cell with BMP4-CM, the early osteogenic differentiation marker ALP activity and ALP staining and later osteogenic differentiation marker calcium deposition were enhanced. However, after treating MEFs cell with DAPT or AddnNotchl and BMP4-conditioned medium （ BMP4-CM）, the early osteogenic differentiation marker ALP activity and ALP staining were inhibited; （3） The expression of essential osteogenic factors （ALP, Runx2, CoUal ） induced by BMP4 were enhanced by DLL1, but were inhibited by DAPT; （4） The protein expression of p-Smadl/5/8 in cell nucleus were enhanced by DLL1, but were inhibited by DAPT; （5） The cell proliferation were promoted by DLL1, but were inhibited by DAPT. Conclusion： Notch signaling promotes BMP4-induced osteogenic differentiation of MSCs by BMP/Smads pathway. At the same time, Notch also enhances cell proliferation during BMP4-induced osteogenic differentiation.

关 键 词：NOTCH信号 骨形态蛋白4 间充质干细胞 成骨分化 

分 类 号：Q819[生物学—生物工程]