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
李珊珊1,2,杨静1,2,张瑾1,2,3
LI Shanshan1,2, YANG Jing1,2, ZHANG Jin1,2,3
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。
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