Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice

Mingjie LU; Hongyan YUAN; Dan XU; Xuelian PENG; Xuqiang ZOU; Bo XIE; Jingwen MAO; Xiujie WEN

doi:10.16016/j.2097-0927.202403078

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Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice

Basic Medicine | 更新时间：2026-01-09

- Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice
- Journal of Army Medical University Vol. 46, Issue 18, Pages: 2069-2080(2024)
- 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：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81970906)
- DOI：10.16016/j.2097-0927.202403078
  CLC： R322.71;R329.24;R394.2
- Received：18 March 2024，
  
  Revised：2024-05-26，
  
  Published：30 September 2024
- 稿件说明：
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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：

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

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