人牙本质基质蛋白1基因启动子在不同细胞中的活性比较和分析
Comparison and Analyss of Human Dentin Matrix Protein 1 Promoter Activity in Three Different Cells
作者：逄键梁;吴补领;张亚庆;赵红萍;刘艳丽
Author：PANG Jian-liang, WU Bu-ling, ZHANG Ya-qing, ZHAO Hong-ping, LIU Yan-li
收稿日期：2006-04-25 年卷（期）页码：2006,24(02):148-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：牙本质基质蛋白1,人牙髓干细胞,成骨细胞,
Key words：dentalmatrixprotein1,humandentalpulpstemcells,osteoblast,
基金项目：
全军“十五”计划基金重点课题资助项目(01Z089)
中文摘要
目的观察并比较不同长度的人牙本质基质蛋白1(Dmp1)基因上游启动子片段在人牙髓干细胞(HDPSC)、成骨细胞(OC)和子宫颈癌上皮细胞株Hela中的启动子活性,为进一步研究Dmp1基因在矿化组织中的特异性转录调控特点和作用机制奠定基础。方法以获得正确序列的2 195 bp Dmp1基因上游启动子重组T载体为模板,通过 PCR方法获得不同长度的Dmp1基因上游启动子片段,将其构建至报告基因载体pGL3-Basic中,瞬时转染至HDP- SC、OC和Hela细胞,荧光素酶检测这些细胞中启动子活性并进行分析。结果成功地获得了6段不同长度的Dmp1 启动子目的片段。酶切鉴定表明不同长度启动子报告基因载体构建成功,不同片段的Dmp1启动子在不同细胞中活性不同,在HDPSC和OC中活性较强,而在Hela细胞中活性最低。在-505--193 bp区和-935--505 bp区可能分别存在HDPSC和OC较为特异的转录调控作用区域点,该区可能包含Dmp1表达的基础调控元件。结论成功克隆了不同长度的Dmp1上游启动子的序列,不同启动子片段在牙髓干细胞和成骨细胞等矿化性细胞中活性较强,并初步确定了Dmp1启动子在矿化性细胞中的基本启动子区域,而在非矿化性细胞Hela细胞中活性较低。
英文摘要
Objective To observe and compare the luciferase activities of different length segments of human dentin matrix protein 1 promoter in human dental pulp stem cells(HDPSC), obteoblasts(OC) and Hela cells. Methods The differentlength disired DNA segments were obtained from 2 0195 bp Dmp1 promoter coloned by PCR method. The amplified promoter segments with different length were cloned into luciferase report gene vector pGL3-Basic, the correct orientation of those inserts was verified by cutting with two different restrict enzymes. The luciferase activity was observed atfer different pGL3-PDmp1 vectors were transfected transiently into those three different-type cells.Results 6Dmp1 promoter segments with different-length wre obtained successfully, and luciferase report gene vectors withdifferent promoter segments were successfully constructed after identified by restriction enzymes cutting. They had different luciferase activities when theywere transfected transiently into HDPSC, and the region of -505---193bp and -935---505bp could be regarded as the specific promoters of Dmp1 promoter for HDPSC and OC respectively, which could include the basic regulatory elements. Conclusion The correct clone of the upstream of human Dmp1 promoter segments with different length had been obtained, and they had strong luciferase activities in HDPSC and OC, but very low in Hela cell. These results will make and important basis for studying mineralized tissue-specifiec transcriptional regulation mechanisms of Dmp1.
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