口腔医学研究

口腔医学研究 ›› 2018, Vol. 34 ›› Issue (1): 18-21.DOI: 10.13701/j.cnki.kqyxyj.2018.01.005

• 口腔干细胞研究 • 上一篇    下一篇

过表达DDIT3对人牙髓干细胞增殖和分化能力影响的研究

王立军*, 刘琼   

  1. 南华大学附属南华医院口腔科 湖南 衡阳 421000
  • 收稿日期:2017-08-15 出版日期:2018-01-28 发布日期:2018-01-26
  • 通讯作者: 王立军,电话:18007479922
  • 作者简介:王立军(1969~ ),男,汉,学士,湖南衡阳人,主治医师,主要从事口腔颌面外科临床治疗工作。
  • 基金资助:
    湖南省教育厅科研项目(编号:14B0104)

Effect of DDIT3 Overexpression on Proliferation and Differentiation of HDPCs

WANG Li-jun*, LIU Qiong   

  1. Department of Stomatology, Nanhua Hospital of University of South China. Hengyang 421001, China
  • Received:2017-08-15 Online:2018-01-28 Published:2018-01-26

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摘要： 目的:通过构建DDIT3过表达质粒,进一步验证过表达DDIT3对人牙髓干细胞(human dental pulp cells,HDPCs)增殖和分化能力的影响。方法:构建带有绿色荧光标记的DDIT3慢病毒载体,分为3组:空白(A组)对照组,绿色荧光对照组(B组),DDIT3过表达组(C组);荧光显微镜观察病毒感染效率;qRT-PCR和Westernblot检测过表达效果;MTT检测三组细胞的增殖能力;ALP、茜素红、vonKossa染色和qRT-PCR检测DDIT3对HDPCs成骨/成牙本质分化能力的影响。结果:荧光显微镜下观察C组绿色荧光表达面积＞90%,qRT-PCR和Westernblot结果表明,C组DDIT3mRNA和蛋白表达明显升高;MTT结果显示,与对照组相比,过表达DDIT3在72 h和96 h可明显抑制HDPCs的增殖;qRT-PCR结果表明,DDIT3过表达可明显增加矿化相关基因OSX,DSPP,DMP1和OCNmRNA表达水平,同时增加DSPP蛋白表达;ALP染色结果表明,3组细胞在成骨/成牙本质分化第7天,均可见明显的ALP染色阳性细胞,但染色面积无明显差异。然而,在成骨/成牙本质分化第14天,茜素红染色和vonKossa染色结果表明,过表达DDIT3可明显增加钙沉积。结论:DDIT3可促进HDPCs晚期的成骨/成牙本质分化。

关键词: DDIT3, HDPCs, 成骨和成脂

Abstract: Objective: To investigate the effect of DDIT3 overexpression on proliferation and osteogenic/odontoblastic differentiation of HDPCs. Methods: DDIT3 overexpression lentiviral construct was confirmed by sequencing. Generation of lentiviral vectors was accomplished using a three-plasmid transfection procedure. Cells were allocated into three groups: HDPCs-WT group, HDPCs-GFP group, and HDPCs-DDIT3-overexpression group. Expression of DDIT3 was quantified by qRT-PCR and Western blot. Cell proliferation and osteogenic/odontoblastic differentiation were detected by MTT, qRT-PCR, ALP staining, Alizarin red staining, and von Kossa staining. Results: A lentiviral vector system was used to efficiently overexpress DDIT3 in primary HDPCs to levels >90%. It was found that DDIT3 mRNA was overexpressed over 38-fold. MTT assay revealed DDIT3 overexpression reduced HDPC proliferation by 72 and 96 hours compared to control groups. DDIT3 overexpression led to no significant difference in ALP staining area after 7 days culture in odontoblastic medium. However, DDIT3 overexpression enhanced calcium deposition by day 14, as examined by alizarin red and von Kossa staining. qRT-PCR results revealed that DDIT3 overexpression did not affect ALP and RUNX2 mRNA levels, however, it significantly increased OSX, DSPP, DMP1, and OCN mRNA levels by day 14. Western blot analysis revealed that DSPP protein levels were higher in the HDPCs-DDIT3-overexpression group. Conclusion: Lipid metabolism related gene DDIT3 may correlate with HDPCs late osteogenic/odontoblastic differentiation.

Key words: DDIT3, HDPCs, Osteogenesis and adipogenesis

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