转录因子Ets-2调控小鼠成釉细胞MMP-20基因表达的研究

Study of Transcription Factor Ets-2 on Regulating the Expression of Matrix Metalloproteinase-20

目的:通过研究Ets-2转录因子对调控小鼠成釉细胞金属基质蛋白酶-20(matrix metalloproteinase-20,MMP-20)基因表达的调控作用,进一步明确Ets-2在釉质发育中的作用。方法:应用免疫组化方法观察出生后5d小鼠切牙成釉细胞中Ets-2的表达;在小鼠成釉细胞中分别转染200ng pcDNA3.1/myc-HisA-Ets-2和Ets-2siRNA后,利用实时定量RT-PCR法检测MMP20基因表达的不同变化;用双荧光素酶报告基因检测系统分析Ets-2对MMP-20启动子突变后转录活性的影响。结果:免疫组化显示Ets-2在成釉细胞中呈阳性表达。实时定量RT-PCR研究发现Ets-2过表达后,MMP-20表达水平显著增加;当Ets-2基因沉默后,MMP-20表达水平则显著下降。双荧光素酶报告基因检测系统检测显示,转染pcDNA3.1/myc-HisA-Ets-2的实验组与对照组相比,MMP-20启动子的转录活性升高;对启动子Ets潜在结合部位进行定点突变后,MMP-20启动子的转录活性显著下降,Ets-2丧失上调MMP-20启动子转录活性的作用。结论:小鼠成釉细胞核中Ets-2可通过MMP-20启动子上Ets潜在结合位点,上调MMP-20基因表达水平。

Objective： To study the significance of Ets-2 in developing dental enamel. Methods： Immunocytochemistry technique were used to observe and analyze the expression of Ets-2 gene in incisors of postnatal 5 d mouse. The 200ng pcDNA3.1/myc-HisA-Ets-2 and the siRNA for Ets-2 mRNA were transfected ALC respectively. Then quantitative real-time PCR was used to measure the different expression of MMP-20. Dual luciferase analysis was used to observe the effects of Ets-2 on the transcriptional activity of MMP20 promoter after mutation of the Ets-2 binding sites. Results： Ets-2 was detected in amelogenesis by immunocytochemistry. By real time PCR, the up-regulation of Ets-2 directly result in a significant higher expression of MMP-20. Contrary to it, knockdown of the transcription factor Ets-2 by siRNA inhibited the MMP20 expression. By the Dual- Luciferase Reporter Assay System, compared with the control group , the transcriptional activity of MMP20 which was transfected with Ets-2 was raised. After mutation of the Ets-2 binding sites, the transcriptional activity of MMP20 had a marked decline. Ets-2 lost the use of regulating the transcriptional activity of MMP--20. Conclusion： Ets-2 up-regulates MMP-20 gene expression via the combination with the Ets-2 binding sites of MMP- 20 in ameloblasts.