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首先利用双荧光素酶基因报告系统分析TGF-β1对MMP20基因启动子转录活性的影响;然后利用染色体免疫共沉淀(Chromatin Immunoprecipitation,ChIP)方法观察Runx2与MMP20特异性结合位点之间的相互作用,并利用基因定点突变和双荧光素酶基因报告系统分析Runx2对MMP20基因启动子转录活性的影响;最后运用小RNA干扰技术使Runx2基因沉默,实时定量RT-PCR技术观察TGF-β1诱导MMP20基因表达的改变。结果:TGF-β1刺激成釉细胞后,MMP20启动子在-87～+23区域转录活性无明显变化外,其他各区域转录活性均增强。
First, the dual luciferase gene reporter system was used to analyze the effect of TGF-β1 on the transcriptional activity of MMP20 promoter. Chromatin Immunoprecipitation (ChIP) was used to observe the interaction between Runx2 and MMP20 specific binding sites The effect of Runx2 on the transcriptional activity of MMP20 gene promoter was analyzed by gene-site-directed mutagenesis and dual luciferase gene reporter system. Runx2 gene silencing was performed by using small RNA interference technique. The expression of MMP20 gene induced by TGF-β1 was observed by real-time quantitative RT-PCR Changes in expression. RESULTS: After ameliorated by ameloblasts, the transcriptional activities of MMP20 promoter in the region of -87 ~ + 23 were not significantly changed, while the transcriptional activities of other regions increased.