小鼠骨硬化蛋白的转录后调控机制

发布时间：2018-01-19 12:10

  本文关键词： 骨硬化蛋白　出处：《山东大学学报(医学版)》2017年03期 　论文类型：期刊论文

【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-REPORTSOST UTR),利用原代培养的小鼠颅骨成骨细胞及M C3T3-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、M C3T3-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(P0.05),而Sost的蛋白水平与对照组相比变化不大(P0.05)。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(P0.05),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(P0.05)。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。
[Abstract]:Objective to construct a luciferase reporter gene vector pMIR-REPORTSOST UTR-containing mouse osteosclerotic protein Sost3 'noncoding region (UTR3). The primary cultured osteoblasts of mouse cranial bone and MC3T3-E1 preosteoblast were used. To explore the posttranscriptional regulation of Sost gene expression during osteogenic differentiation. Methods Osteogenic C57BL / 6 mouse primary calvarial osteoblasts and MC3T3-E1 cells were induced by osteogenesis. The relative expression levels of mRNA and protein of Sost gene were detected respectively. PCR was used to clone the 3UTR region of Sost gene. Linked to luciferase reporter gene vector pMIR-REPORT. Name the build recombinant vector pMIR-REPORT-SOST UTR. change the pMIR-REPORT-SOST. UTR and pMIR-REPORT were transfected into osteoblasts. MC3T3-E1 cells and NIH3T3 mouse embryonic fibroblasts. According to the transfection plasmid, the cells were divided into pMIR-REPORT-SOST UTR group and pMIR-REPORT group. Results the mRNA levels of Sost in osteoblasts and MC3T3-E1 cells of osteogenic mice were significantly higher than those of the control group (P < 0.05). P0.05). Compared with the control group, the protein level of Sost did not change significantly (P 0.05). It was found in the osteoblasts and MC3T3-E1 cells of the mouse skull. The relative level of luciferase expression in pMIR-REPORT-SOST UTR transfection group was lower than that in pMIR-REPORT transfection group (P 0.05). However, there was no significant difference in luciferase expression between pMIR-REPORT transfection group and pMIR-REPORT-SOST UTR transfection group in NIH3T3 cells. Conclusion pMIR-REPORT-SOST UTR was successfully constructed. Moreover, the 3 Sost region of Sost gene is involved in cell-specific post-transcriptional regulation.
【作者单位】： 山东大学口腔医学院;山东省口腔组织再生重点实验室;山东大学口腔医院牙体牙髓科;
【基金】：国家自然科学基金(81100757)
【分类号】：R68
【正文快照】： 网络出版地址:http://www.cnki.net/kcms/detail/37.1390.R.20170302.1110.014.html骨硬化蛋白(sclerostin,SOST)是由Sost基因编码的含有213个氨基酸的糖蛋白,它可以通过与LRP5/6受体结合而抑制经典的Wnt信号通路,从而抑制成骨细胞的增殖和分化[1-2]。Sost基因敲除小鼠的骨量，

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