Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells

Wei FENG; Zhengsong XIE; Cen LUO; Xiaolin TU

doi:10.16016/j.1000-5404.202001002

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Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells

Basic Medicine | 更新时间：2026-01-09

- Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells
- Journal of Third Military Medical University Vol. 42, Issue 9, Pages: 899-907(2020)
- 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：
- 基金信息：
  
  the China-Guangdong Joint Fund of National Natural Science Foundation(U1601220)
- DOI：10.16016/j.1000-5404.202001002
  CLC： R322.71;R329.2;R394-33
- Received：01 January 2020，
  
  Revised：2020-01-26，
  
  Published：15 May 2020
- 稿件说明：
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Wei FENG, Zhengsong XIE, Cen LUO, et al. Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells[J]. Journal of Third Military Medical University, 2020, 42(9): 899-907.
DOI：

Wei FENG, Zhengsong XIE, Cen LUO, et al. Construction of MLO-Y4 cells with overexpression of Delta-like genes and their effects on osteogenic differentiation of bone marrow stromal cells[J]. Journal of Third Military Medical University, 2020, 42(9): 899-907. DOI： 10.16016/j.1000-5404.202001002.

摘要

目的

建立稳定表达外源Dll1、Dll3、Dll4基因的MLO-Y4骨细胞株，研究骨细胞Notch信号Delta-like配体Dll1、Dll3、Dll4对骨髓基质细胞成骨分化的影响。

方法

用过表达Dll1、Dll3、Dll4基因的重组慢病毒液感染MLO-Y4样骨细胞，嘌呤霉素筛选分别稳定表达Dll1、Dll3及Dll4基因的MLO-Y4骨细胞株。采用实时荧光定量PCR(real time fluorescence quantitative，qPCR)对筛选出来的MLO-Y4骨细胞株中Dll1、Dll3及Dll4基因的表达进行鉴定。将3种细胞株分别与野生型小鼠骨髓基质细胞(bone marrow stromal cells，BMSCs)进行共培养，采用碱性磷酸酶(ALP)染色及ALP定量检测共培养3 d ALP活性变化；qPCR检测共培养3 d成骨相关标志基因及Notch信号靶基因的表达水平，在使用Notch信号抑制剂DAPT后检测上述指标。

结果

与阴性对照组相比，Dll1、Dll3及Dll4转染组中Dll1、Dll3及Dll4 mRNA的水平显著增加(

0.05)。共培养3 d后，骨细胞中Dll4基因的过表达促进了BMSC的ALP活性，与阴性对照组相比有统计学差异(

0.05)。qPCR结果显示，MLO-Y4-Dll4组的ALP、Runx2、OPN及CollagenⅠ成骨相关标志基因的表达均有所升高(

0.05)，Notch信号靶基因Hey1、HeyL、Hes1及Hes7的表达也有所升高(

0.05)。而在加入DAPT后，MLO-Y4-Dll4组的ALP活性下降，成骨相关标志基因及Notch信号靶基因的表达有所下降(

0.05)。

结论

建立了稳定表达外源Dll1、Dll3或Dll4基因的MLO-Y4骨细胞株，在骨细胞中过表达Dll4能促进骨髓基质细胞的早期成骨分化，经典Notch信号通路在其中可能发挥了重要作用。

Abstract

Objective

To construct the MLO-Y4 cell strains stably expressing exogenous genes of ligands for Notch receptors Delta like-1 (Dll1)

Dll3

and Dll4 and determine the effects of these ligands on osteogenic differentiation of bone marrow stromal cells.

Methods

MLO-Y4 cells were infected with recombinant lentiviral vectors carrying Dll1

Dll3 and Dll4 genes. Puromycin was used to choose the MLO-Y4 cells with stable expression of these genes. Then

real-time fluorescence quantitative (qPCR) was applied to identify the expression of above genes in the selected MLO-Y4 cells. The 3 cell strains were respectively co-cultured with wild-type mouse bone marrow stromal cells (BMSCs). In 3 d after co-culture

alkaline phosphatase (ALP) staining and ALP quantitative detection were used to detect changes in ALP activity

and qPCR was employed to detect the expression levels of osteogenesis-related markers and target genes of Notch signal. The above indicators were measured again after DAPT

a Notch signaling inhibitor was added in the culture medium.

Results

Compared with the blank group and the negative control

the mRNA levels of Dll1

Dll3

and Dll4 were significantly increased in the corresponding transfection cells (

0.05). After 3 days of co-culture

the overexpression of Dll4 gene in MLO-Y4 cells promoted the ALP activity in the BMSCs

which was significantly stronger than that of the negative control group (

0.05). qPCR results showed that the expression levels of osteogenesis-related marker genes

such as ALP

Runx2

OPN and CollagenⅠ in the MLO-Y4-Dll4 group were all risen (

0.05)

and so were those of Notch signaling target genes

Hey1

HeyL

Hes1 and Hes7 (

0.05). However

the addition of DAPT inhibited the ALP activity in the MLO-Y4-Dll4 group

and decreased the expression levels of above osteogenesis-related markers and Notch signaling target genes (

0.05).

Conclusion

MLO-Y4 cell strains stably expressing the exogenous genes Dll1

Dll3 and Dll4 are established successfully. Overexpression of Dll4 in osteocytes can promote early osteogenic differentiation of BMSCs

and the classic Notch signaling pathway may play an important role in this process.

关键词

Keywords

references

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