体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响

谢映春; 涂小林

doi:10.16016/j.1000-5404.202001048

您当前的位置：

首页 >

文章列表页 >

体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响

基础医学 | 更新时间：2026-01-09

- 体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响
- Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro
- 第三军医大学学报 2020年42卷第9期页码：891-898
- 作者机构：
  
  400016 重庆，重庆医科大学生命科学研究院骨发育与再生平台
- 作者简介：
  
  [涂小林，E-mail：xiaolint@hotmail.comTU Xiaolin, E-mail:xiaolint@hotmail.com
- 基金信息：
  
  国家自然科学基金面上项目(81672118)
- DOI：10.16016/j.1000-5404.202001048
  中图分类号： R322.71;R329.2;R394-33
- 收稿：2020-01-07，
  
  修回：2020-02-23，
  
  纸质出版：2020-05-15
- 稿件说明：
移动端阅览
谢映春, 涂小林. 体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响[J]. 第三军医大学学报, 2020,42(9):891-898.

Yingchun XIE, Xiaolin TU. Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro[J]. Journal of Third Military Medical University, 2020, 42(9): 891-898.
谢映春, 涂小林. 体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响[J]. 第三军医大学学报, 2020,42(9):891-898. DOI： 10.16016/j.1000-5404.202001048.

Yingchun XIE, Xiaolin TU. Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro[J]. Journal of Third Military Medical University, 2020, 42(9): 891-898. DOI： 10.16016/j.1000-5404.202001048.

摘要

目的

探究体外激活骨细胞Notch信号对骨髓基质细胞(bone marrow stromal cells

BMSCs)成骨分化的影响。

方法

设置Cre重组腺

病毒(Ad-Cre)及对照腺病毒GFP(Ad-GFP)，分别转染Rosa

Notch

小鼠骨细胞后与野生C57BL/6小鼠BMSCs共培养为Ad-Cre组及Ad-GFP组，设置仅BMSCs为Blank组。碱性磷酸酶染色及生化定量测定碱性磷酸酶(ALP)；qPCR检测转染后骨细胞Notch信号靶基因及共培养产物成骨相关标志物、Notch信号配体及成血管相关标志物，茜素红染色检测成骨诱导21 d钙盐沉积水平。

结果

Ad-Cre成功激活Rosa

Notch

小鼠骨细胞Notch信号，ALP染色及生化定量结果显示Ad-Cre组ALP表达相较Ad-GFP组及Blank组显著降低(

0.05)，Ad-GFP组与Blank组间无统计学差异；qPCR结果显示Ad-Cre组共培养产物成骨标志物ALP、核心结合因子a1(Runx2)、特异性骨转化因子(Ostrix)及Notch配体Dll4 mRNA转录水平相较Ad-GFP组及Blank组显著降低(

0.05)，Jag1 mRNA转录水平显著降低(

0.05)；血管内皮生长因子(VEGF)、低氧诱导因子(hypoxia-inducible factor 1

，HIF1

)、血小板内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1

CD31/PECAM-1)及内皮粘蛋白(endomucin

EMCN)mRNA转录水平相较Ad-GFP组及Blank组显著增高(

0.05)；茜素红染色结果显示Ad-Cre组在成骨诱导培养基作用21 d后钙盐沉积水平相较Ad-GFP组及Blank组显著降低。

结论

体外激活骨细胞Notch信号抑制骨髓基质细胞成骨分化。

Abstract

Objective

To investigate the effect and underlying mechanism of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells (BMSCs).

Methods

The osteocytes derived from Rosa

Notch

mice were infected with recombinant adenovirus expressing Cre or GFP respectively

and then the obtained cells were co-cultured with BMSCs isolated from wild-type C57BL/6 mice

and named as Ad-Cre group and Ad-GFP group

with a BMSCs group without any treatment as blank control. Alkaline phosphatase (ALP) staining and detection of ALP relative activity were used to measure the expression of ALP. The mRNA expression levels of the target genes in osteocyte of Rosa

Notch

and ligands of Notch signaling

osteogenic markers

and angiogenic makers in the co-cultured product were detected by qPCR. Alizarin red staining was applied to test the matrix mineralization on day 21.

Results

Infection of

recombinant adenovirus Ad-Cre could successfully activate Notch signaling. ALP staining and detection of ALP activity showed that the expression level of ALP was significantly lower in the Ad-Cre group than the Ad-GFP group and Blank group (

0.05)

and there was no statistical difference in the level between the latter 2 groups. The results of qPCR indicated that the mRNA levels of osteogenic makers ALP

Osterix

Runx2 and Notch ligand Dll4 were deceased significantly

while those of Jag1

VEGF

hypoxia-inducible factor 1

(HIF1

)

platelet endothelial cell adhesion molecule-1 (CD31/PECAM-1)

and endomucin (EMCN) were statistically increased in the Ad-Cre group when compared with the Ad-GFP group and Blank group (

0.05). Alizarin red staining displayed that the Ad-Cre group had more calcium deposition in 21 d after co-culture than the Ad-GFP group and blank group (

0.05).

Conclusion

Activating Notch signaling in osteocytes inhibits osteogenic differentiation of BMSCs

in vitro.

关键词

Keywords

references

BI P P, KUANG S H. Notch signaling as a novel regulator of metabolism[J]. Trends Endocrinol Metab, 2015, 26(5): 248-255. DOI:10.1016/j.tem.2015.02.006.

CAMPBELL D P, CHRYSOSTOMOU E, DOETZLHOFER A. Canonical Notch signaling plays an instructive role in auditory supporting cell development[J]. Sci Rep, 2016, 6: 19484. DOI:10.1038/srep19484.

CHEUNG L, LE TISSIER P, GOLDSMITH S G, et al . NOTCH activity differentially affects alternative cell fate acquisition and maintenance[J ] . Elife, 2018, 7: e33318. DOI:10.7554/eLife.33318.

COLOMBO M, THVMMLER K, MIRANDOLA L, et al . Notch signaling drives multiple myeloma induced osteoclastogenesis[J ] . Oncotarget, 2014, 5(21): 10393-10406. DOI:10.18632/oncotarget.2084.

KUSHWAH R, GUEZGUEZ B, LEE J B, et al . Pleiotropic roles of Notch signaling in normal, malignant, and developmental hematopoiesis in the human[J ] . EMBO Rep, 2014, 1 5(11): 1128-1138. DOI:10.15252/embr.201438842.

WAHI K, BOCHTER M S, COLE S E. The many roles of Notch signaling during vertebrate somitogenesis[J]. Semin Cell Dev Biol, 2016, 49: 68-75. DOI:10.1016/j.semcdb.2014.11.010.

COLOMBO M, GALLETTI S, GARAVELLI S, et al . Notch signaling deregulation in multiple myeloma: a rational molecular target[J ] . Oncotarget, 2015, 6(29): 26826-26840. DOI:10.18632/oncotarget.5025.

ZANOTTI S, CANALIS E. Notch signaling and the skeleton[J]. Endocr Rev, 2016, 37(3): 223-253. DOI:10.1210/er.2016-1002.

NG A H, BAHT G S, ALMAN B A, et al . Bone marrow stress decreases osteogenic progenitors[J ] . Calcif Tissue Int, 2015, 97(5): 476-486. DOI:10.1007/s00223-015-0032-3.

BONEWALD L F. The role of the osteocyte in bone and nonbone disease[J]. Endocrinol Metab Clin N Am, 2017, 46(1): 1-18. DOI:10.1016/j.ecl.2016.09.003.

ANSARI N, HO P W, CRIMEEN-IRWIN B, et al . Autocrine and pa racrine regulation of the murine skeleton by osteocyte-derived parathyroid hormone-related protein[J ] . J Bone Miner Res, 2018, 33(1): 137-153. DOI:10.1002/jbmr.3291.

TOKARZ D, MARTINS J S, PETIT E T, et al . Hormonal regulation of osteocyte perilacunar and canalicular remodeling in the hyp mouse model of X-linked hypophosphatemia[J ] . J Bone Miner Res, 2018, 33(3): 499-509. DOI:10.1002/jbmr.3327.

DELGADO-CALLE J, ANDERSON J, CREGOR M D, et al . Bidirectional notch signaling and osteocyte-derived factors in the bone marrow microenvironment promote tumor cell proliferation and bone destruction in multiple myeloma[J ] . Cancer Res, 2016, 76(5): 1089-1100. DOI:10.1158/0008-5472.CAN-15-1703.

JÄHN K, KELKAR S, ZHAO H, et al . Osteocytes acidify their microenvironment in response to PTHrP in vitro and in lactating mice in vivo [J ] . J Bone Miner Res, 2017, 32(8): 1761-1772. DOI:10.1002/jbmr.3167..

LIU W, WANG Z Y, YANG J, et al . Osteocyte TSC1 promotes sclerostin secretion to restrain osteogenesis in mice[J ] . Open Biol, 2019, 9(5): 180262. DOI:10.1098/rsob.180262.

NAKASHIMA T, HAYASHI M, FUKUNAGA T, et al . Evidence for osteocyte regulation of bone homeostasis through RANKL expression[J ] . Nat Med, 2011, 17(10): 1231-1234. DOI:10.1038/nm.2452.

CANALIS E, ADAMS D J, BOSKEY A, et al . Notch signaling in osteocytes differentially regulates cancellous and cortical bone remodeling[J ] . J Biol Chem, 2013, 288(35): 25614-25625. DOI:10.1074/jbc.M113.470492.

XU Y, SHU B, TIAN Y, et al . Notch activation promotes osteoblast mineralization by inhibition of apoptosis[J ] . J Cell Physiol, 2018, 233(10): 6921-6928. DOI:10.1002/jcp.26592.

CANALIS E, BRIDGEWATER D, SCHILLING L, et al . Canonical Notch activation in osteocytes causes osteopetrosis[J ] . Am J Physiol Endocrinol Metab, 2016, 310(2): E171-E182. DOI:10.1152/ajpendo.00395.2015.

ZANOTTI S, CANALIS E. Activation of Nfatc2 in osteoblasts causes osteopenia[J]. J Cell Physiol, 2015, 230(7): 1689-1695. DOI:10.1002/jcp.24928.

ZANOTTI S, SMERDEL-RAMOYA A, STADMEYER L, et al . Notch inhibits osteoblast differentiation and cau ses osteopenia[J ] . Endocrinology, 2008, 149(8): 3890-3899. DOI:10.1210/en.2008-0140.

DEREGOWSKI V, GAZZERRO E, PRIEST L, et al . Notch 1 overexpression inhibits osteoblastogenesis by suppressing wnt/β-catenin but not bone morphogenetic protein signaling[J ] . J Biol Chem, 2006, 281(10): 6203-6210. DOI:10.1074/jbc.m508370200.

SHAH K M, STERN M M, STERN A R, et al . Osteocyte isolation and culture methods[J ] . Bonekey Rep, 2016, 5: 838. DOI:10.1038/bonekey.2016.65.

MARIDAS D E, RENDINA-RUEDY E, LE P T, et al . Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice[J ] . J Vis Exp, 2018(131): 56750. DOI:10.3791/56750.

ISO T, KEDES L, HAMAMORI Y. HES and HERP families: multiple effectors of the Notch signaling pathway[J]. J Cell Physiol, 2003, 194(3): 237-255. DOI:10.1002/jcp.10208.

KRATZER R F, KREPPEL F. Production, purification, and titration of first-generation adenovirus vectors[J]. Methods Mol Biol, 2017, 1654: 377-388. DOI:10.1007/978-1-4939-7231-9_28.

ENGIN F, YAO Z Q, YANG T, et al . Dimorphic effects of Notch signaling in bone homeostasis[J ] . Nat Med, 2008, 14(3): 299-305. DOI:10.1038/nm1712.

HILTON M J, TU X L, WU X M, et al . Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation[J ] . Nat Med, 2008, 14(3): 306-314. DOI:10.1038/nm1716.

SHAO J, ZHOU Y H, XIAO Y. The regulatory roles of Notch in osteocyte differentiation via the crosstalk with canonical Wnt pathways during the transition of osteoblasts to osteocytes[J ] . Bone, 2018, 108: 165-178. DOI:10.1016/j.bone.2018.01.010.

TU X L, DELGADO-CALLE J, CONDON K W, et al . Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone[J ] . Proc Natl Acad Sci USA, 2015, 112(5): E478-E486. DOI:10.1073/pnas.1409857112.

任磊, 代光明, 林枭, 等.骨细胞Wnt/β-Catenin通过Notch信号促进BMSCs成骨分化[J].中国骨质疏松杂志, 2018, 24(5): 600-605.

REN L, DAI G M, LIN X, et al . Wnt/β-Catenin induces osteoblastic differentiation of BMCS via Notch signaling pathway by osteocytes[J ] . Chin J Osteoporos, 2018 , 24(5): 600-605..

WANG N, LIU W, TAN T, et al . Notch signaling negatively regulates BMP9-induced osteogenic differentiation of mesenchymal progenitor cells by inhibiting JunB expression[J ] . Oncotarget, 2017, 8(65): 109661-109674. DOI:10.18632/oncotarget.22763.

BIGAS A, ROBERT-MORENO A, ESPINOSA L. The Notch pathway in the developing hematopoietic system[J]. Int J Dev Biol, 2010, 54(6/7): 1175-1188. DOI:10.1387/ijdb.093049ab.

FRASER H M, HASTINGS J M, ALLAN D, et al . Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary[J ] . Endocrinology, 2012, 153(4): 1972-1983. DOI:10.1210/en.2011-1688.

BENEDITO R, ROCA C, SÖRENSEN I, et al . The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis[J ] . Cell, 2009, 137(6): 1124-1135. DOI:10.1016/j.cell.2009.03.025.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

Notch信号介导骨细胞过表达BMP4促进骨髓基质细胞成骨分化

TGF-βⅡ型受体小分子抑制剂ITD-1对骨髓基质细胞成骨分化和血管形成的作用

过表达Notch配体Delta-like基因的MLO-Y4骨细胞株的建立及其对骨髓基质细胞成骨分化的作用

新型二甲双胍碳点促进炎症微环境下人牙周膜干细胞成骨分化

骨化三醇对脂多糖诱导猪主动脉瓣膜间质细胞成骨分化的影响