TGF-β1调控基质金属蛋白酶-20(MMP-20)启动子转录活性的研究  

The regulation of TGF-beta on the transcription activity of matrix metalloproteinase-20(MMP-20)promoter

作　　者：韩婷婷[1] 孙岩[1] 张娟娟[1] 刘晓影[1] 官秀梅[1] 高玉光[1] 

机构地区：[1]潍坊医学院口腔医学院口腔医学研究所,山东潍坊261042

出　　处：《牙体牙髓牙周病学杂志》2009年第5期251-255,共5页Chinese Journal of Conservative Dentistry

基　　金：国家自然科学基金(30672316);山东省自然科学基金(Y2006C105);山东省教育厅重点项目(J06L22)

摘　　要：目的:克隆MMP-20基因启动子序列,分析转化生长因子-β1对其转录活性的影响。方法:检索并分析MMP-20基因翻译起始点上游序列。利用PCR从小鼠基因组上扩增长度为2852bp的启动子序列,并与pMD18-T克隆载体连接。经酶切反应初步鉴定后,以pMD18-T-MMP-20为模板,利用PCR方法获得带酶切位点的MMP-20启动子片段(2842bp),并与pGL3-Basic载体连接。pGL3-MMP-20瞬时转染小鼠成釉细胞系(ALC)过夜后,在培养基中加入1ng/mLTGF-β1,12h后检测荧光素酶的活性。结果:经酶切反应和测序结果证实MMP-20启动子成功插入pGL3-Basic质粒。与pGL3-Basic质粒转染相比较,转染pGL3-MMP-20后荧光素酶活性明显增强;培养基中加入TGF-β1能促使pGL3-MMP-20荧光素酶活性进一步增强。结论:外源性MMP-20启动子在成釉细胞中被激活;TGF-β1能增强pGL3-MMP-20荧光素酶活性,提示釉质发育过程中,TGF-β1具有调节MMP-20基因表达的生物学功能。AIM ： To clone the promoter sequences of MMP - 20 gene, and to analyze the effects of TGF - betal on the transcription activity of the promoter. METHODS ： The upstream regulation sequences of MMP - 20 gene were retrieved and analyzed. The 2852bp fragment was amplified from the genomic DNA of mouse by PCR, and inserted into pMD18 -T vector. The pMD18 -T - MMP -20 plasmid was digested with restriction endonucleases enzyme to verify that the amplified fragment was correct. The 2842bp fragment with enzyme - cutting sites of MMP - 20 promoter was amplified and inserted into pGL3 - basic vector, and verified by enzyme digestion and sequencing. The pGL3 - MMP- 20 vector was transiently transfeeted into ameloblast- lineage cells （ALC） overnight, then 1 ng/mL TGF- betal was added to the culture medium for 12hrs. The assay was performed to examine the luciferase activity. RESULTS ： The enzyme digestion and sequencing results confirmed that the 2842bp fragment of MMP - 20 was inserted into the pGL3 - basic plasmid. Compared with the pGL3 - basic plasmid transfection, the transfection of the pGL3 - MMP - 20 plasmid showed a strong luciferase activity. After adding TGF - betal to the medium, the luciferase activity of pGL3 - MMP - 20 vector was further promoted. CONCLUSION ： TGF - betal can enhance the transcription activity of MMP- 20 gene promoter,which suggestes that TGF -betal has a role in regulating MMP- 20 gene expression during enamel development.

关 键 词：转化生长因子-Β1 基质金属蛋白酶-20 启动子 双荧光素酶报告系统 

分 类 号：R780.2[医药卫生—口腔医学]