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山东大学学报（医学版） ›› 2017, Vol. 55 ›› Issue (3): 43-48.doi: 10.6040/j.issn.1671-7554.0.2016.1068

• 基础医学 • 上一篇    下一篇

小鼠骨硬化蛋白的转录后调控机制

李珊珊1,2,杨静1,2,张瑾1,2,3   

  1. 1.山东大学口腔医学院;2.山东省口腔组织再生重点实验室;3.山东大学口腔医院牙体牙髓科, 山东 济南 250012
  • 收稿日期:2016-08-29 出版日期:2017-03-10 发布日期:2017-03-10
  • 通讯作者: 张瑾. E-mail: teadenzj@sdu.edu.cn E-mail:teadenzj@sdu.edu.cn
  • 基金资助:
    国家自然科学基金(81100757)

Post-transcriptional regulation mechanism of sclerostin in mice

LI Shanshan1,2, YANG Jing1,2, ZHANG Jin1,2,3   

  1. 1.School of Stomatology, Shandong University;
    2. Shandong Provincial Key Laboratory of Oral Tissue Regeneration;
    3. Department of Endodontics, School and Hospital of Stomatology, Shandong University, Jinan 250012, Shandong, China
  • Received:2016-08-29 Online:2017-03-10 Published:2017-03-10

摘要： 目的 构建含有小鼠骨硬化蛋白(Sost)基因3'非编码区(UTR)的荧光素酶报告基因载体(pMIR-REPORT-SOST UTR),利用原代培养的小鼠颅骨成骨细胞及MC3T3-E1小鼠前成骨细胞系,探究在成骨分化过程中Sost基因表达所受到的转录后调控作用。 方法 成骨诱导C57BL/6小鼠原代颅骨成骨细胞及MC3T3-E1细胞,分别检测各细胞中Sost基因mRNA和蛋白的相对表达水平。采用PCR克隆Sost基因3'UTR区,连接到荧光素酶报告基因载体pMIR-REPORT上,命名构建重组载体为pMIR-REPORT-SOST UTR。将pMIR-REPORT-SOST UTR和pMIR-REPORT转染至颅骨成骨细胞、MC3T3-E1细胞及NIH3T3小鼠胚胎成纤维细胞中,根据转染质粒将细胞分为pMIR-REPORT-SOST UTR组和pMIR-REPORT组,检测各组细胞中荧光素酶的相对表达水平。 结果 成骨诱导小鼠颅骨成骨细胞及MC3T3-E1细胞Sost的mRNA水平较对照组均明显升高(P<0.05),而Sost的蛋白水平与对照组相比变化不大(P>0.05)。在小鼠颅骨成骨细胞及MC3T3-E1细胞中,pMIR-REPORT-SOST UTR转染组较pMIR-REPORT转染组的荧光素酶相对表达水平均有所下降(P<0.05),而在NIH3T3细胞中pMIR-REPORT转染组和pMIR-REPORT-SOST UTR转染组的荧光素酶相对表达水平差异无统计学意义(P>0.05)。 结论 成功构建了pMIR-REPORT-SOST UTR,且Sost基因的3'UTR区参与了具有细胞特异性的转录后调控。

关键词: Wnt信号, 骨硬化蛋白, 成骨分化, 转录后调控, 3'非编码区

Abstract: Objective To investigate whether the expression of Sost gene was post-transcriptionally regulated in primary osteoblasts and MC3T3-E1 murine pre-osteoblast cells. Methods Osteogenic differentiation was induced in MC3T3-E1 cells and primary calvarial osteoblasts were isolated from C57BL/6 mice. The mRNA and protein levels of Sost gene in these cells were evaluated respectively. The 3'UTR of mouse Sost gene was amplified and subcloned into the SacI restriction site of pMIR-REPORT miRNA expression reporter, and the resulted plasmid was named as pMIR-REPORT-SOST 3' untranslated regions(UTR). Calvarial osteoblasts, MC3T3-E1 cells and NIH3T3 cells were then transfected with pMIR-REPORT-SOST UTR and pMIR-REPORT, respectively. Results Sost mRNA levels in calvarial osteoblasts and MC3T3-E1 cells were higher than control groups(P<0.05)during osteogenic differentiation. In contrast, the protein levels of Sost gene had no significant difference(P>0.05)with control group in calvarial osteoblasts and MC3T3-E1 cells. Relative luciferase levels transfected with pMIR-REPORT-SOST UTR had dramatically decline with transfected with pMIR-REPORT, respectively(P<0.05). In contrast, relative luciferase levels had no statistically significant differences between cells transfected with pMIR-REPORT-SOST UTR and pMIR-REPORT in NIH3T3 cells(P>0.05). Conclusion pMIR-REPORT-SOST UTR was successfully generated, and the expression of Sost gene was post-transcriptionally regulated via the 3'UTR.

Key words: Sclerostin, Osteogenic differentiation, Wnt signaling, Post-transcriptional regulation, 3'Untranslated regions(UTR)

中图分类号: 

  • R574
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