激活转录因子-2调控小鼠成釉细胞基质金属蛋白酶-20分子机制的初步研究

Preliminary study of the molecular mechanisms of activating transcription factor-2 regulating matrix metalloproteinase-20 in mice ameloblasts

目的:通过克隆激活转录因子-2(ATF-2)基因以及真核表达载体的构建,初步研究ATF-2对小鼠成釉细胞基质金属蛋白酶-20(MMP-20)基因表达的影响。方法:根据引物设计原则设计ATF-2基因逆转录-聚合酶链反应(PCR)引物,从小鼠成釉细胞中提取总RNA逆转录所得c DNA为模板,进行PCR法扩增。得出含有HindⅢ和xhol酶切位点的ATF-2目的基因连接到pc DNA 3.1/myc-His A真核表达载体上;用重组质粒转染小鼠MMP-20基因,观察其对MMP-20基因转录活性的影响。结果:经过PCR引物扩增得到1 463 bp基因片段,将获得的重组质粒pc DNA 3.1/myc-His A-ATF-2双酶切分析鉴定,测序结果与Gen Bank登录基因序列完全一致;双荧光素酶结果显示ATF-2可以促进MMP-20启动子的表达(P<0.01),且在-825^+23(848 bp)启动子区段促进作用最明显。结论:成功实现了ATF-2基因克隆及真核表达载体的构建,并发现ATF-2对MMP-20启动子区域活性表达起正向调节作用。

Objective： To clone the activating transcription factor-2（ ATF-2） gene,then construct the recombinant eukaryotic expression vector with ATF-2,and explore the effects of ATF-2 on the gene expression of matrix metalloproteinase-20（ MMP-20） in mice ameloblasts. Methods： The reverse transcription PCR primers of ATF-2 were designed. The total RNA from the mice ameloblasts was extracted to obtain c DNA for PCR amplification. The ATF-2 gene containing Hind Ⅲ and xhol restriction sites was inserted into the eukaryotic expression vector（ pc DNA 3. 1 / myc-His A）. The effects of ATF-2 on the transcriptional activity of MMP-20 was observed after transfecting the recombinant plasmid. Results： One thousand four hundred and sixty-three bp gene fragment was amplified through PCR. The recombinant eukaryotic expression vector with ATF-2（ pc DNA 3. 1 / myc-His A-ATF-2） was successful constructed by the verification of dual enzyme digestion and sequencing. The luciferase test showed that ATF-2 gene could promote the expression of MMP-20 promoter（ P〈0. 01）. Conclusions： The recombinant pc DNA 3. 1 / myc-His A-ATF-2 expression vector is successful constructed.ATF-2 can positive regulate the promoter activity of MMP-20.