萝卜硫素促进骨髓间充质干细胞向成骨细胞分化的分子机制研究

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萝卜硫素促进骨髓间充质干细胞向成骨细胞分化的分子机制研究

基础研究 | 更新时间：2024-01-22

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- 萝卜硫素促进骨髓间充质干细胞向成骨细胞分化的分子机制研究
- Molecular mechanism of sulforaphane promoting the differentiation of bone marrow stem cells into osteoblasts
- 局解手术学杂志 2024年33卷第1期页码：24-29
- 作者机构：
  
  1.武汉科技大学附属孝感医院骨关节与运动医学科，湖北孝感 432100
  2.武汉科技大学医学院，湖北武汉 430065
- 作者简介：
  
  张宇博，E-mail：1049756476@qq.com
- 基金信息：
  
  湖北省卫生健康委员会科研项目(WJ2021M044)
- DOI：10.11659/jjssx.02E023123
  中图分类号： R394.2
- 纸质出版日期：2024-01-25，
  
  收稿日期：2023-02-23，
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张铮,韩佳雯,彭龙龙等.萝卜硫素促进骨髓间充质干细胞向成骨细胞分化的分子机制研究[J].局解手术学杂志,2024,33(01):24-29.

ZHANG Zheng,HAN Jia-wen,PENG Long-long,et al.Molecular mechanism of sulforaphane promoting the differentiation of bone marrow stem cells into osteoblasts[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(01):24-29.
张铮,韩佳雯,彭龙龙等.萝卜硫素促进骨髓间充质干细胞向成骨细胞分化的分子机制研究[J].局解手术学杂志,2024,33(01):24-29. DOI： 10.11659/jjssx.02E023123.

ZHANG Zheng,HAN Jia-wen,PENG Long-long,et al.Molecular mechanism of sulforaphane promoting the differentiation of bone marrow stem cells into osteoblasts[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(01):24-29. DOI： 10.11659/jjssx.02E023123.

摘要

目的

2

探讨萝卜硫素（Sul）促进骨髓间充质干细胞（BMSCs）向成骨细胞分化的分子机制。

方法

2

将BMSCs分为对照组（不做任何处理）、诱导组（诱导成骨分化）、诱导+Sul组（诱导成骨分化并加入40 μmol/L Sul）；分别向BMSCs转染腺病毒-shRNA-Mock、-shRNA-TET1、-shRNA-TET2、-shRNA-TET3，作为shRNA-Mock组、shRNA-TET1组、shRNA-TET2组、shRNA-TET3组；BMSCs于含成骨分化诱导培养液及添加40 μmol/L Sul的细胞培养液中培养，然后转染腺病毒-shRNA-TET1、-shRNA-TET2、-shRNA-TET3、-shRNA-Mock，作为诱导+Sul+shRNA-TET1组、诱导+Sul+shRNA-TET2组、诱导+Sul+shRNA-TET3组、诱导+Sul+shRNA-Mock组。qPCR法和Western blot法测定BMSCs向成骨细胞分化后Runx2 mRNA和蛋白的表达水平。染色质免疫共沉淀（ChIP）实验测定BMSCs向成骨细胞分化后，与Histone H3结合的Runx2启动子区DNA含量。HpaⅡ酶和MspⅠ酶消化联合qPCR法测定BMSCs向成骨细胞分化后Runx2启动子区的甲基化水平。茜素红染色测定BMSCs向成骨细胞分化的程度。

结果

2

与诱导组相比，诱导+Sul组Runx2 mRNA和蛋白表达水平明显上升（

P

<

0.05），与Histone H3结合的Runx2启动子区DNA含量增高（

P

<

0.05），Runx2启动子区的甲基化水平降低（

P

<

0.05），茜素红染色评分升高（

P

<

0.05）。与诱导+Sul组相比，诱导+Sul+shRNA-TET1组细胞内与Histone H3结合的Runx2启动子区DNA含量降低（

P

<

0.05），Runx2启动子区的甲基化水平升高（

P

<

0.05），茜素红染色评分明显降低（

P

<

0.05）；而诱导+Sul+shRNA-TET2组、诱导+Sul+shRNA-TET3组及诱导+Sul+shRNA-Mock组则无明显变化（

P

>

0.05）。

结论

2

Sul能够通过TET1促进Runx2启动子区DNA去甲基化，促进BMSCs向成骨细胞分化。

Abstract

Objective

2

To investigate the molecular mechanism of sulforaphane （Sul） promoting bone marrow stem cells （BMSCs） differentiating into osteoblasts.

Methods

2

BMSCs were divided into the control group （without any treatment），induction group （induction of osteogenic differentiation），and induction+Sul group （induction of osteogenic differentiation with the addition of 40 μmol/L of Sul）. The adenovirus-shRNA-Mock，-shRNA-TET1，-shRNA-TET2，and -shRNA-TET3 were transfected into BMSCs as the shRNA-Mock group，shRNA-TET1 group，shRNA-TET2 group，and shRNA-TET3 group. BMSCs were cultured in cell culture medium containing osteogenic differentiation induction medium and 40 μmol/L of Sul，and then transfected with adenovirus-shRNA-TET1，-shRNA-TET2，-shRNA-TET3，and -shRNA-Mock as the induction+Sul+shRNA-TET1 group，induction+Sul+shRNA-TET2 group，induction+Sul+shRNA-TET3 group，and induction +Sul+shRNA-Mock group. The mRNA and protein expression levels of Runx2 after BMSCs differentiated into osteoblasts were determined by qPCR and Western blot. The DNA content of Runx2 promoter region bound to Histone H3 after BMSCs differentiated into osteoblasts was determined by chromatin immunocoprecipitation （ChIP）. The methylation level of Runx2 promoter region of BMSCs differentiated into osteoblasts was determined by HpaⅡenzyme and MspⅠenzyme digestion combined with qPCR. The degree of BMSCs differentiated into osteoblasts was determined by alizarin red staining.

Results

2

Compared with the induction group，the mRNA and protein expression levels of Runx2 in the induction+Sul group were significantly increased （

P

<

0.05）； the content of DNA in the Runx2 promoter region bound to Histone H3 was increased （

P

<

0.05），the methylation level of Runx2 promoter region was reduced （

P

<

0.05），and the alizarin red staining score was elevated （

P

<

0.05）. Compared with the induction+Sul group，the content of DNA in the Runx2 promoter region bound to Histone H3 in the induction+Sul+shRNA-TET1 group was decreased （

P

<

0.05），the methylation level of Runx2 promoter region was increased （

P

<

0.05），and the alizarin red staining score was decreased （

P

<

0.05）. While there was no significant change among the induction+Sul+shRNA-TET2 group，induction+Sul+shRNA-TET3 group，induction+Sul+shRNA-Mock group （

P

>

0.05）.

Conclusion

2

Sul can promote the differentiation of BMSCs into osteoblasts through promoting DNA demethylation of Runx2 promoter region by TET1.

关键词

骨髓间充质干细胞成骨分化萝卜硫素Runx2表观遗传学DNA甲基化

Keywords

bone marrow stem cellsosteoblasts differentiationsulforaphaneRunx2epigeneticsDNA methylation

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