Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用

陆明洁; 袁洪燕; 徐丹; 彭雪莲; 邹续强; 谢波; 毛靖雯; 温秀杰

doi:10.16016/j.2097-0927.202403078

您当前的位置：

首页 >

文章列表页 >

Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用

基础医学 | 更新时间：2026-01-09

- Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用
- Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice
- 陆军军医大学学报 2024年46卷第18期页码：2069-2080
- 作者机构：
  
  646000 四川泸州，西南医科大学附属口腔医院正畸科，口颌面修复重建和再生泸州市重点实验室，西南医科大学口腔医学研究所
- 作者简介：
  
  [温秀杰, E-mail: wenxiujie@tom.comWEN Xiujie, E-mail: wenxiujie@tom.com
- 基金信息：
  
  国家自然科学基金面上项目(81970906)
- DOI：10.16016/j.2097-0927.202403078
  中图分类号： R322.71;R329.24;R394.2
- 收稿：2024-03-18，
  
  修回：2024-05-26，
  
  纸质出版：2024-09-30
- 稿件说明：
移动端阅览
陆明洁, 袁洪燕, 徐丹, 等. Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用[J]. 陆军军医大学学报, 2024,46(18):2069-2080.

Mingjie LU, Hongyan YUAN, Dan XU, et al. Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice[J]. Journal of Army Medical University, 2024, 46(18): 2069-2080.
陆明洁, 袁洪燕, 徐丹, 等. Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用[J]. 陆军军医大学学报, 2024,46(18):2069-2080. DOI： 10.16016/j.2097-0927.202403078.

Mingjie LU, Hongyan YUAN, Dan XU, et al. Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice[J]. Journal of Army Medical University, 2024, 46(18): 2069-2080. DOI： 10.16016/j.2097-0927.202403078.

摘要

目的

探讨黑色素瘤相关抗原D1(melanoma associated antigen D1

Mage-D1)对小鼠股骨骨量和对大鼠骨髓间充质干细胞(bone mesenchymal stromal cells

BMSCs)矿化能力的影响及潜在分子机制。

方法

以雌性Mage-D1基因敲除杂合子小鼠和雄性野生型(wide type

WT)小鼠作为亲本小鼠，繁育Mage-D1基因敲除纯合子(Mage-D1 knock out

Mage-D1 KO)小鼠。PCR和琼脂糖凝胶电泳鉴定雄性Mage-D1基因敲除纯合子小鼠和同窝雄性野生型小鼠，micro-CT扫描用于分析小鼠股骨骨量，ELISA和化学法用于检测小鼠血清钙离子、磷离子、降钙素和甲状旁腺素水平。原代培养BMSCs并进行流式细胞术鉴定，免疫荧光染色观察Mage-D1在BMSCs中的表达。构建Mage-D1沉默慢病毒感染BMSCs，分为阴性对照组(sh-NC)和沉默组(sh-Mage-D1)；细胞划痕实验检测BMSCs的迁移能力，流式细胞术和CCK-8检测BMSCs周期变化和增殖能力；矿化诱导后行碱性磷酸酶染色、茜素红染色；RT-qPCR和Western blot检测ALP、Runx2、Col1表达水平；RT-qPCR检测矿化相关基因

NTR

及

MSX

1以探讨潜在机制。

结果

与野生型小鼠相比，Mage-D1敲除纯合子小鼠股骨皮质骨厚度减小、皮质骨矿物含量降低、松质骨骨矿物含量降低、骨小梁数减少、松质骨骨表面密度降低、骨小梁分离度增大(

0.05)；敲除Mage-D1后，小鼠血钙、血磷、降钙素和甲状旁腺素水平无明显变化。Mage-D1表达于整个BMSCs内，在细胞核及胞核周围区域高表达。与sh-NC相比，sh-Mage-D1组细胞增殖能力降低(

0.01)，细胞迁移能力增强(

0.01)；矿化诱导后，sh-Mage-D1组ALP、Runx2、Col1基因(

0.05)和蛋白(

0.01)表达下降，碱性磷酸酶染色和茜素红染色更浅；sh-Mage-D1组细胞中

NTR

、

Msx

1表达水平比sh-NC组低。

结论

Mage-D

1基因敲除可明显降低小鼠股骨骨量。Mage-D1可促进BMSCs增殖、抑制其迁移，正向调控其体外矿化，p75NTR-Dlx1/Msx1信号轴可能参与Mage-D1对骨代谢活动的调控。

Abstract

Objective

To investigate the effect of melanoma associated antigen D1 (Mage-D1) on mouse femoral bone mass and mineralization ability of mouse bone marrow mesenchymal cells (BMSCs) and its potential molecular mechanism.

Methods

Female Mage-D1 gene knockout heterozygous mice and male wild-type (WT) mice were subjected as parent mice to breed Mage-D1 gene knockout homozygous (Mage-D1 KO) mice. PCR and agarose gel electrophoresis were used to identify male Mage-D1 knockout (Mage-D1 KO) mice and littermate male wild-type (WT) mice. Micro-CT scanning was performed to observe mouse femoral bone mass

and ELISA and chemical assay were employed to detect serum levels of calcium

phosphorus

calcitonin

and parathyroid hormone in mice. After primary cultured BMSCs were identified with flow cytometry

immunofluorescence staining was utilized to detect the expression of Mage-D1 in BMSCs. BMSCs were infected by Mage-D1 silencing lentivirus

and then the cells were divided into negative control group (sh-NC) and silencing group (sh-Mage-D1). Cell scratch assay was conducted to detect the migration ability of BMSCs

and flow cytometry and CCK-8 assay were conducted to detect the cycle change and proliferation ability of BMSCs. After mineralization induction

alkaline phosphatase (ALP) staining and alizarin red staining were performed; RT-qPCR and Western blotting were used to measure the expression levels of ALP

Runx2 and Col1. RT-qPCR was used to detect mineralization-related genes p75NTR and Msx1.

Results

Compared with the WT mice

the femoral cortical bone thickness

cortical bone mineral content

cancellous bone mineral content

trabecular number

and cancellous bone surface density were decreased

and trabecular separation was increased in the Mage-D1 knockout homozygous mice (

0.05). There were no significant changes in the serum levels of calcium

phosphorus

calcitonin and parathyroid hormone in mice after Mage-D1 knockout. Mage-D1 was expressed in the whole BMSCs and was highly expressed in the nucleus and perinuclear regions. Compared with the sh-NC BMSCs

the sh-Mage-D1 group had decreased proliferation ability (

0.01)

enhanced migration ability (

0.01)

and decreased expression of ALP

Runx2 and Col1 genes (

0.05) and protein (

0.01) after mineralization induction

milder ALP and aliz

arin red stain

and lower expression levels of p75NTR and Msx1.

Conclusion

Mage-D1 knockout can significantly reduce femur bone mass in mice. It can promote the proliferation and inhibit migration of BMSCs

and positively regulate their mineralization

in vitro

and the p75NTR-Dlx1/Msx1 signaling axis may be involved in the regulation of bone metabolism by Mage-D1.

关键词

Keywords

references

GAO Y G, PATIL S, JIA J X. The development of molecular biology of osteoporosis[J]. Int J Mol Sci, 2021, 22(15): 8182. DOI: 10.3390/ijms22158182.

PÕLD M, ZHOU J, CHEN G L, et al . Identification of a new, unorthodox member of the MAGE gene family[J ] . Genomics, 1999, 59(2): 161-167. DOI: 10.1006/geno.1999.5870..

DU Q, ZHANG Y, TIAN X X, et al . MAGE-D1 inhibits proliferation, migration and invasion of human breast cancer cells[J ] . Oncol Rep, 2009, 22(3): 659-665. DOI: 10.3892/or_00000486.

REDDY E M, CHETTIAR S T, KAUR N, et al . Dlxin-1, a MAGE family protein, induces accelerated neurite outgrowth and cell survival by enhanced and early activation of MEK and Akt signalling pathways in PC12 cells[J ] . Exp Cell Res, 2010, 316(14): 2220-2236. DOI: 10.1016/j.yexcr.2010.05.030.

MOURI A, NODA Y, WATANABE K, et al . The roles of MAGE-D1 in the neuronal functions and pathology of the central nervous system[J ] . Rev Neurosci, 2013, 24(1): 61-70. DOI: 10.1515/revneuro-2012-0069.

SASAKI A, MASUDA Y, IWAI K, et al . A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein, Dlxin-1[J ] . J Biol Chem, 2002, 277(25): 22541-22546. DOI: 10.1074/jbc.M109728200.

BERTRAND M J, KENCHAPPA R S, ANDRIEU D, et al . NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo [J ] . Cell Death Differ, 2008, 15(12): 1921-1929. DOI: 10.1038/cdd.2008.127..

LIU M, XU L J, MA X, et al . MAGED1 is a negative reg ulator of bone remodeling in mice[J ] . Am J Pathol, 2015, 185(10): 2653-2667. DOI: 10.1016/j.ajpath.2015.06.017.

LIU H X, ZHANG X, YANG Q X, et al . Knockout of NRAGE promotes autophagy-related gene expression and the periodontitis process in mice[J ] . Oral Dis, 2021, 27(3): 589-599. DOI: 10.1111/odi.13575.

WU Q, LI J, SONG P P, et al . Knockdown of NRAGE induces odontogenic differentiation by activating NF-κB signaling in mouse odontoblast-like cells[J ] . Connect Tissue Res, 2019, 60(2): 71-84. DOI: 10.1080/03008207.2018.1439484.

MASUDA Y, SASAKI A, SHIBUYA H, et al . Dlxin-1, a novel protein that binds Dlx5 and regulates its transcriptional function[J ] . J Biol Chem, 2001, 276(7): 5331-5338. DOI: 10.1074/jbc.M008590200.

ICHIDA F, NISHIMURA R, HATA, et al . Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation[J ] . J Biol Chem, 2004, 279(32): 34015-34022. DOI: 10.1074/jbc.M403621200.

MAEDA K, KOBAYASHI Y, UDAGAWA N, et al . Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances ost eoclastogenesis[J ] . Nat Med, 2012, 18(3): 405-412. DOI: 10.1038/nm.2653.

DOMBRET C, NGUYEN T, SCHAKMAN O, et al . Loss of Maged1 results in obesity, deficits of social interactions, impaired sexual behavior and severe alteration of mature oxytocin production in the hypothalamus[J ] . Hum Mol Genet, 2012, 21(21): 4703-4717. DOI: 10.1093/hmg/dds310.

ARNOLD A, DENNISON E, KOVACS C S, et al . Hormonal regulation of biomineralization[J ] . Nat Rev Endocrinol, 2021, 17(5): 261-275. DOI: 10.1038/s41574-021-00477-2.

HSIAO C Y, CHEN T H, CHU T H, et al . Calcitonin induces bone formation by increasing expression of Wnt10b in osteoclasts in ovariectomy-induced osteoporotic rats[J ] . Front Endocrinol, 2020, 11: 613. DOI: 10.3389/fendo.2020.00613.

CHEN Y X, JIN L, XUE B, et al . NRAGE induces β-catenin/Arm O-GlcNAcylation and negatively regulates Wnt signaling[J ] . Biochem Biophys Res Commun, 2017, 487(2): 433-437. DOI: 10.1016/j.bbrc.2017.04.080.

ABOU NADER Z, ESPÉLI M, BALABANIAN K, et al . Culture, expansion and differentiation of mouse bone-derived me senchymal stromal cells[J ] . Methods Mol Biol, 2021, 2308: 35-46. DOI: 10.1007/978-1-0716-1425-9_3.

LIN Z Y, HE H B, WANG M, et al . MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate[J ] . Cell Prolif, 2019, 52(6): e12688. DOI: 10.1111/cpr.12688.

LI M, YU X, LUO Y T, et al . Dynamic expression of Mage-D1 in rat dental germs and potential role in mineralization of ectomesenchymal stem cells[J ] . Sci Rep, 2022, 12(1): 22615. DOI: 10.1038/s41598-022-27197-5.

GORDEEVA O, GORDEEV A, KHAYDUKOV S. Expression dynamics of Mage family genes during self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells[J ] . Oncotarget, 2019, 10(35): 3248-3266. DOI: 10.18632/oncotarget.26933.

YOUNG A T, MOORE R B, MURRAY A G, et al . Assessment of different transfection parameters in efficiency optimization[J ] . Cell Transplant, 2004, 13(2): 179-185.

MUNKÁCSY G, SZTUPINSZKI Z, HERMAN P, et al . Validation of RNAi silencing efficiency using gene array data shows 18.5% failure rate across 429 independent experiments[J ] . Mol Ther Nucleic Acids, 2016, 5(9): e366. DOI: 10.1038/mtna.2016.66.

OWENT A, ARONOW M, SHALHOUB V, et al . Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix[J ] . J Cell Physiol, 1990, 143(3): 420-430. DOI: 10.1002/jcp.1041430304.

LEEM N, HWANG H S, OH S H, et al . Elevated extracellular calcium ions promote proliferation and migration of mesenchymal stem cells via increasing osteopontin expression[J ] . Exp Mol Med, 2018, 50(11): 1-16. DOI: 10.1038/s12276-018-0170-6.

MIAO M M, ZHANG Y, WANG X P, et al . The miRNA-144-5p/IRS1/AKT axis regulates the migration, proliferation, and mineralization of osteoblasts: a mechanism of bone repair in diabetic osteoporosis[J ] . Cell Biol Int, 2022, 46(12): 2220-2231. DOI: 10.1002/cbin.11913.

QI S, WU Q, MA J, et al . Effects of neurotrophin receptor-mediated MAGE homology on proliferation and odontoblastic differentiation of mouse dental pulp cells[J ] . Cell Prolif, 2015, 48(2): 221-230. DOI: 10.1111/cpr.12171.

LIU L J, MICHOWSKI W, KOLODZIEJCZYK A, et al . The cell cycle in stem cell proliferation, pluripotency and differentiation[J ] . Nat Cell Biol, 2019, 21(9): 1060-1067. DOI: 10.1038/s41556-019-0384-4.

SHEN W G, XUE Q Y, WU Y D, et al . Melanoma-associated antigen family protein-D1 regulation of tumor cell migration, adhesion to endothelium, and actin structures reorganization in response to hypoxic stress[J ] . Cell Commun Adhes, 2007, 14(1): 21-31. DOI: 10.1080/15419060701224948.

CHU C S, XUE B, TU C, et al . NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo [J ] . Cancer Lett, 2007, 250(2): 268-275. DOI: 10.1016/j.canlet.2006.10.020..

SALHOTRA A, SHAHH N, LEVI B, et al . Mechanisms of bone development and repair[J ] . Nat Rev Mol Cell Biol, 2020, 21(11): 696-711. DOI: 10.1038/s41580-020-00279-w.

KOMORI T, YAGI H, NOMURA S, et al . Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J ] . Cell, 1997, 89(5): 755-764. DOI: 10.1016/s0092-8674(00)80258-5.

MASCI M, WANG M, IMBERT L, et al . Bone mineral properties in growing Col1a2(+/G610C) mice, an animal model of osteogenesis imperfecta[J ] . Bone, 2016, 87: 120-129. DOI: 10.1016/j.bone.2016.04.011.

CALABRESE G, BENNETT B J, OROZCO L, et al . Systems genetic analysis of osteoblast-lineage cells[J ] . PLoS Genet, 2012, 8(12): e1003150. DOI: 10.1371/journal.pgen.1003150.

YANG K, WANG Y Y, JU Y X, et al . p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells[J ] . Cell Prolif, 2017, 50(1): e12290. DOI: 10.1111/cpr.12290.

ZHAO M Z, WANG Y Y, LI G, et al . The role and potential mechanism of p75NTR in mineralization via in vivo p75NTR knockout mice and in vitro ectomesenchymal stem cells[J ] . Cell Prolif, 2020, 53(2): e12758. DOI: 10.1111/cpr.12758..

罗玉婷, 杨正艳, 李蒙, 等. Mage-D1与活化后的p75神经营养因子受体结合可正向调节大鼠外胚间充质干细胞的矿化[J]. 南方医科大学学报, 2021, 41(10): 1547-1553. DOI: 10.12122/j.issn.1673-4254.2021.10.14.

LUO Y T, YANG Z Y, LI M, et al . Mage-D1 binding to activated p75NTR positively regulates mineralization of rat ectomesenchymal stem cells in vitro [J ] . J South Med Univ, 2021, 41(10): 1547-1553. DOI: 10.12122/j.issn.1673-4254.2021.10.14..

浏览量

156

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

Alg13 突变致痫小鼠的繁育及鉴定

黑色素瘤相关抗原D1在小鼠牙发育过程中的动态表达规律与调控作用研究

盘状结构域受体1对实验性结肠炎小鼠肠道炎症及纤维化的影响

Gpr183基因敲除小鼠的构建及其在急性肝损伤中功能初步分析

Fscb/Cabyr双基因敲除对雄鼠繁殖能力的影响