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

doi:10.13523/j.cb.20170407

中国生物工程杂志

2017, Vol. 37

Issue (4): 48-55 DOI: 10.13523/j.cb.20170407

研究报告

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

曹俊杰, 李爱芳, 卫亚琳, 廉静, 唐敏

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

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

CAO Jun-jie, LI Ai-fang, WEI Ya-lin, LIAN Jing, TANG Min

Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing 400016, China

全文: PDF(1535 KB) HTML

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（AddnNotch1）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dnNotch1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。

关键词： Notch信号; 间充质干细胞; 成骨分化; 骨形态蛋白4

Abstract: 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 AddnNotch1 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, Col1a1) induced by BMP4 were detected by RT-PCR; (4) The protein expression of p-Smad1/5/8 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 AddnNotch1 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, Col1a1) induced by BMP4 were enhanced by DLL1, but were inhibited by DAPT; (4) The protein expression of p-Smad1/5/8 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.

Key words: Bone morphogenetic protein 4 Osteogenic differentiation Notch signaling Mesenchymal stem cells

收稿日期: 2016-08-12 出版日期: 2017-04-25

ZTFLH:

Q819

基金资助: 重庆市自然科学基金（CSTC2012jjA10004;KJ130305）资助项目

通讯作者: 唐敏 E-mail: Catom@126.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	曹俊杰
	卫亚琳
	廉静
	李爱芳
	唐敏

引用本文:

曹俊杰, 李爱芳, 卫亚琳, 廉静, 唐敏. 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[J]. 中国生物工程杂志, 2017, 37(4): 48-55.

CAO Jun-jie, LI Ai-fang, WEI Ya-lin, LIAN Jing, TANG Min. Role of Notch Signaling Pathway in Bone Morphogenetic Protein 4-induced Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells and Its Mechanism. China Biotechnology, 2017, 37(4): 48-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170407 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I4/48

[1] Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147.
[2] Rastegar F, Shenaq D, Huang J, et al. Mesenchymal stem cells:Molecular characteristics and clinical applications. World Journal of Stem Cells, 2010, 2(4):67-80.
[3] Caplan A I, Bruder S P. Mesenchymal stem cells:building blocks for molecular medicine in the 21st century. Trends in Molecular Medicine, 2001, 7(6):259-264.
[4] Garreta E, Genove E, Borros S, et al. Osteogenic differentiation of mouse embryonic stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold. Tissue Engineering, 2006, 12(8):2215-2227.
[5] Lengner C J, Lepper C, van Wijnen A J, et al. Primary mouse embryonic fibroblasts:a model of mesenchymal cartilage formation. Journal of Cellular Physiology, 2004, 200(3):327-333.
[6] Saeed H, Taipaleenmaki H, Aldahmash A M, et al. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC). Stem Cell Reviews, 2012, 8(2):318-328.
[7] Peng Y, Kang Q, Cheng H, et al. Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. Journal of Cellular Biochemistry, 2003, 90(6):1149-1165.
[8] Penton A L, Leonard L D, Spinner N B. Notch signaling in human development and disease. Seminars in Cell & Developmental Biology, 2012, 23(4):450-457.
[9] Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch. Journal of Bone and Mineral Research:The Official Journal of The American Society for Bone and Mineral Research, 2002, 17(2):231-239.
[10] Canalis E. Notch signaling in osteoblasts. Science Signaling, 2008, 1(17):e17.
[11] 廉静, 杨伦韵, 曹俊杰, 等. DLL1在骨形态形成蛋白9诱导骨髓间充质干细胞成骨分化中的作用. 第三军医大学学报, 2016, 38(1):38-43. Lian J, Yang L Y, Cao J J, et al. Delta-like 1 promotes osteogenic differentiation of mesenchymal stem cells induced by BMP9. Journal of Third Military Medical University, 2016, 38(1):38-43.
[12] Viale-Bouroncle S, Gosau M, Morsczeck C. NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells. Biochemical and Biophysical Research Communications, 2014, 443(2):500-504.
[13] 杨伦韵, 张晓艳, 廉静, 等. Notch1在BMP9诱导iMEF细胞成骨分化中的作用.第三军医大学学报, 2015,37(1):39-45. Yang L Y, Zhang X Y, Lian J, et al. Role of Notch1 signaling in the process of BMP9-induced osteogenetic differentiation of immortalized mouse embryonic fibroblasts. Journal of Third Military Medical University, 2015,37(1):39-45.
[14] Varnum-Finney B, Wu L, Yu M, et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. Journal of Cell Science, 2000, 113(23):4313-4318.
[15] Cordle J, Redfieldz C, Stacey M, et al. Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity. The Journal of Biological Chemistry, 2008, 283(17):11785-11793.
[16] Zanotti S, Canalis E. Notch Signaling and the skeleton. Endocrine Reviews, 2016, 37(3):223-253
[17] Kopan R, Ilagan M X. The canonical Notch signaling pathway:unfolding the activation mechanism. Cell, 2009, 137(2):216-233.
[18] Luu H H, Song W X, Luo X, et al. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. Journal of Orthopaedic Research:Official Publication of the Orthopaedic Research Society, 2007, 25(5):665-677.
[19] Jane J. Ectopic osteogenesis using adenoviral bone morphogenetic protein (BMP)-4 and BMP-6 gene transfer. Molecular Therapy, 2002, 6(4):464-470.
[20] Buas M F, Kadesch T. Regulation of skeletal myogenesis by Notch. Experimental Cell Research,2010, 316(18):3028-3033.
[21] Dahlqvist C, Blokzijl A, Chapman G, et al. Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development, 2003, 130(24):6089-6099.

[1]	王宇轩,陈婷,张永亮. MiR-148生物学功能研究进展^*[J]. 中国生物工程杂志, 2021, 41(7): 74-80.
[2]	李开秀,司维. 间充质干细胞来源的外泌体治疗炎症性肠病研究进展^*[J]. 中国生物工程杂志, 2021, 41(7): 66-73.
[3]	赵久梅,王哲,李学英. 调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的作用^*[J]. 中国生物工程杂志, 2021, 41(10): 62-72.
[4]	苑亚坤,刘广洋,刘拥军,谢亚芳,吴昊. 间充质干细胞基础研究与临床转化的中美比较[J]. 中国生物工程杂志, 2020, 40(4): 97-107.
[5]	陈利军,屈晶晶,项春生. 间充质干细胞在2019新型冠状病毒肺炎(COVID-19)中的治疗潜能、临床研究与应用前景^*[J]. 中国生物工程杂志, 2020, 40(11): 43-55.
[6]	朱永朝,陶金,任萌萌,熊燃,何亚琴,周瑜,卢震辉,杜勇,杨芝红. 自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 ^*[J]. 中国生物工程杂志, 2019, 39(9): 62-67.
[7]	朱颖,范梦恬,李具琼,陈彬,张盟浩,吴静红,施琼. 趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究 ^*[J]. 中国生物工程杂志, 2019, 39(8): 7-16.
[8]	程瑜,施琼,安利钦,范梦恬,皇改改,翁亚光. **BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化 ^***[J]. 中国生物工程杂志, 2019, 39(5): 63-71.
[9]	施文雯,张蕾. 力学微环境影响间充质干细胞分化的研究现状 ^*[J]. 中国生物工程杂志, 2018, 38(8): 76-83.
[10]	郑妍,姚欢,杨珂. SFRP5抑制BMP9诱导人脐带间充质干细胞成骨分化的实验研究 ^*[J]. 中国生物工程杂志, 2018, 38(7): 7-13.
[11]	袁雅红, 赵珊珊, 王小莉, 腾智平, 李东升, 曾毅. HIV-1 Tat蛋白抑制骨髓间充质干细胞的造血支持功能[J]. 中国生物工程杂志, 2017, 37(6): 1-8.
[12]	刘红霞, 施琼, 周一青, 安利钦, 严树涓, 张汝益, 翁亚光. 过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化[J]. 中国生物工程杂志, 2017, 37(5): 9-18.
[13]	刘晓骅, 吉彩霞, 徐丽, 董超群, 罗进勇. Hmox1促进BMP9诱导C3H10T1/2细胞向成骨分化[J]. 中国生物工程杂志, 2017, 37(4): 33-39.
[14]	吉彩霞, 刘晓骅, 徐丽, 董超群, 罗进勇. Runx1促进BMP9诱导的间充质干细胞MEFs的成骨分化[J]. 中国生物工程杂志, 2017, 37(3): 10-17.
[15]	陈文杰, 汪建样, 殷明, 殷嫦嫦. 人脐带间充质干细胞抗肿瘤机制的研究进展[J]. 中国生物工程杂志, 2017, 37(3): 78-82.

Viewed

Full text

Abstract

Cited

Shared

Discussed