华西口腔医学杂志 ›› 2012, Vol. 30 ›› Issue (5): 468-473.doi: 10.3969/j.issn.1000-1182.2012.05.005

• 基础研究 • 上一篇    下一篇

血管内皮生长因子和转化生长因子β1基因调控人根尖乳头细胞矿化相关因子的研究

杨海兵1，2 韩萱1 杨琳1 王燕1   

  1. 1.山东大学口腔医院牙体牙髓病科； 山东省口腔生物医学重点实验室， 济南250012；
    2.南京医科大学附属常州市第二人民医院口腔科， 常州213003
  • 收稿日期:2011-11-21 修回日期:2012-06-22 出版日期:2012-10-01 发布日期:2012-10-01
  • 通讯作者: 王燕，Tel：0531-88382623
  • 作者简介:杨海兵（1986—），男，江苏人，住院医师，硕士
  • 基金资助:

    山东省科技发展计划基金资助项目（2010G0020230）；山东大学自主创新基金自然科学类专项交叉学科培育基金资助项目（2011JC019）

Study of vascular endothelial growth factor and transforming growth factor β1 genes regulate the mineralrelated genes in human cells from apical papilla

Yang Haibing1，2, Han Xuan1, Yang Lin1, Wang Yan1.   

  1. 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
  • Received:2011-11-21 Revised:2012-06-22 Online:2012-10-01 Published:2012-10-01

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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&beta, 1, 转染, 根尖乳头细胞, 矿化作用

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）, osteocalcin（OCN）, dentin matrix protein 1（DMP1） were detected by Real-Time polymerase chain reaction（PCR）. Results Cloned VEGF165 gene sequences and inserted into expression vector of the VEGF165 sequences showed 100% homology related to the sequence in GenBank database. VEGF165 and TGFβ1 mRNA were upregulated after transfection. The expression of DSPP mRNA were significantly increased in each experiment group（P＜0.05）. The expression of OCN mRNA were increased significantly in the group transfected with pcDNA3.1hisA-TGFβ1 and transfected with two plasmids（P＜0.05）. The expression of BSP mRNA were not varying（P＞0.05）, while no expression of DMP1 mRNA in each experiment group. Conclusion The recombinant eucaryotic expression vector of pcDNA3.1hisA-VEGF165 was constructed successfully. VEGF165 and TGFβ1 can induce the expression of most mineral-related genes and they may play a key role during the differentiation of human cells from apical papilla.

Key words: vascular endothelial growth factor； gene clone； transforming growth factor &beta, 1； transfection； cells from apical papilla； mineralization

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