C3H10T 1/2小鼠胚胎成纤维细胞Dlxin 1基因表达及骨形态发生蛋白2的调控机制  

Dlxin 1 gene expression and bone morphogenetic protein 2 regulation mechanism in C3H10T 1/2 mouse embryo fibroblasts

作　　者：万阳[1] 杨松海 潘秋辉[3] 温传俊[4] 孙奋勇[1] 马纪[1] 洪岸[1] 

机构地区：[1]暨南大学生物工程研究所,广东省广州市510632 [2]广东韶关市铁路医院,广东省韶关市512023 [3]中山大学附属孙逸仙纪念医院医学研究中心,广东省广州市510120 [4]南京师范大学生命科学学院分子医学生物技术江苏省重点实验室,江苏省南京市210097

出　　处：《中国组织工程研究与临床康复》2009年第33期6518-6522,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:Dlxin1可与成骨相关转录因子Dlx、Msx家族成员相互作用调节其转录活性,骨形态发生蛋白2可诱导小鼠胚胎成纤维细胞(C3H10T1/2)向成骨细胞分化。目的:对C3H10T1/2小鼠胚胎成纤维细胞中Dlxin1基因的表达及骨形态发生蛋白2调控机制进行初探。设计、时间及地点:细胞分子水平实验,于2007-03/10在暨南大学生物工程研究所实验室完成。材料:C3H10T1/2细胞为ATCC产品,骨形态发生蛋白2为自产,DH5α为自备。方法:用荧光定量PCR(qRT-PCR)检测骨形态发生蛋白2对基因表达的影响,用双荧光报告载体系统搜寻Dlxin1的上游启动子元件,用凝胶迁移实验(EMSA)对其精确定位。主要观察指标:①细胞中Dlxin1基因mRNA的表达情况。②pGL3-Dlxin1系列缺失突变报告载体荧光活性及启动子片段与C3H10T1/2细胞核蛋白的结合能力检测。结果:在转录水平上骨形态发生蛋白2可以诱导Dlxin1基因表达上调;Dlxin1启动子基础活性和骨形态发生蛋白2诱导活性调控区域均位于转录起始位点上游-943～-728bp之间;EMSA证实,-758～-748bp这一片段是调控核心区域。结论:转录因子通过与Dlxin1启动子上游TATAbox结合来调控Dlxin1基因的转录。BACKGROUND： Dlxin 1 has been shown to have important functions in regulating the transcriptional activity of several osteoblast transcription factors, such as Dlx and Msx family members. Bone morphogenetic protein 2 （BMP2） can induce mouse embryo fibroblasts （MEFs, C3H10T 1/2） differentiating into osteoblasts. OBJECTIVE： To make a pilot study on the Dlxin 1 gene expression and the BMP2 regulation mechanisms in C3H10T 1/2 MEFs. DESIGN, TIME AND SETTING： A cellular and molecular level study was performed in the Institute of Bioengineering, Jinan University, from March to October in 2007. MATERIALS： C3H10T1/2 cell lines were purchased from American Type Culture Collection （ATCC）. BMP2 was produced and DH5α was saved by our laboratory.METHODS： The effect of BMP2 on Dlxin 1 gene expression was detected by quantitative Reverse Transcription-PolymeraseChain Reaction Analysis （qRT-PCR）. The upstream promoter element （UPE） of Dlxin 1 gene was indentified by Dual-LuciferaseReporter Assay System, and then its exact location was determined with Electrophoretic Mobility Shift Assay （EMSA）.MAIN OUTCOME MEASURES： ①The Dlxin 1 gene mRNA expression in cells. ②Luciferase activity of pGL3-Dlxin 1 deletionmutation reporter vectors and the binding capacity of Dlxin 1 promoter fragments to C3H10T 1/2 cell nuclear proteins.RESULTS： On the transcription level, BMP2 could upregulate the expression of Dlxin 1 in C3H10T 1/2 MEFs; Both basic activeregions and active regulation regions （under induction with BMP 2） of Dlxin 1 promoter existed between -943 bp and -728 bpupstream of the transcriptional start site. Further analysis with EMSA indicated that the genomic fragments between -758 bp and-748 bp upstream of the transcriptional start site played a vital role in the regulation of Dlxin 1 transcription.CONCLUSION： Transcription factors regulate Dlxin 1 transcription through its interaction with the TATA box in the upstream ofDlxin 1 gene promoter.

关 键 词：Dlxin 1 启动子 骨形态发生蛋白2 成骨 C3H10T 1/2 

分 类 号：R349.83[医药卫生—基础医学]