血管内皮生长因子与转化生长因子β1基因调控人根尖乳头细胞矿化相关因子探究.doc

血管内皮生长因子与转化生长因子β1基因调控人根尖乳头细胞矿化相关因子探究[摘要] 目的 克隆人血管内皮生长因子（VEGF）异构体中的VEGF165，构建真核表达载体，探讨过表达VEGF165和转化生长因子β1（TGFβ1）对人根尖乳头细胞矿化相关因子的影响。方法 提取人脐静脉内皮细胞系ECV304总RNA，采用逆转录聚合酶链反应（RT-PCR）方法扩增VEGF165基因，插入pcDNA3.1hisA，构建重组质粒pcDNA3.1hisA-VEGF165，经酶切及测序验证正确后，将其和pcDNA3.1hisA-TGFβ1转染人根尖乳头细胞，采用实时定量聚合酶链反应检测转染效率和骨涎蛋白（BSP）、牙本质涎磷蛋白（DSPP）、骨钙素（OCN）、牙本质基质蛋白1（DMP1）的表达。结果 插入表达载体的VEGF165基因序列与GenBank数据库中的序列具有100%同源性；转染后VEGF165及TGFβ1 mRNA显著增高；各实验组DSPP mRNA的表达均升高（P＜0.05），实验2组和实验3组的OCN mRNA升高（P＜0.05），各组间BSP mRNA的表达差异无统计学意义（P＞0.05），DMP1 mRNA均未见表达。结论 成功构建VEGF165真核表达载体，VEGF165和TGFβ1 均能促进人根尖乳头细胞多种矿化因子的表达，与根尖乳头细胞的分化相关。 [关键词] 血管内皮生长因子； 基因克隆； 转化生长因子β1； 转染； 根尖乳头细胞； 矿化作用 [中图分类号] Q 786 [文献标志码] A [doi] 10.3969/j.issn.1000-1182.2012.05.005 Study of vascular endothelial growth factor and transforming growth factor β1 genes regulate the mineral-related genes in human cells from apical papilla Yang Haibing1，2, Han Xuan1, Yang Lin1, Wang Yan1. （1. Dept. of Endodontics, School of Stomatology, Shandong University; Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan 250012, China; 2. Dept. of Stomatology, The Affiliated Hospital of Nanjing Medical University, Changzhou No.2 People’s Hospital, Changzhou 213003, China） [Abstract] Objective To clone the VEGF165 gene and to construct eucaryotic expression vector, investigate the effect of overexpressed VEGF165 and transforming growth factor β1（TGFβ1） on the mineral-related genes in human cells from apical papilla. Methods Total RNA of ECV304 cell was extracted. The VEGF165 gene was amplified by reverse transcription-polymerase chain reaction（RT-PCR）, and then was subcloned into eucaryotic expression vector pcDNA3.1hisA to construct the recombinant vector pcDNA3.1hisA-VEGF165. After being identified by digestion and DNA sequencing, pcDNA3.1hisA-VEGF165 and pcDNA3.1hisA-TGFβ1 were transfected into human cells from apical papilla. Then the efficiency of gene transfection and the expression of bone sialoprotein（BSP）, dentin sialophosphoprotein （DSPP）,