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

摘要

Objective To clone the VEGF165 gene and to construct eucaryotie 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 eucaryotie 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-TGFpi 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 expres-sion 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. ConclusionThe recombinant eucaryotie expression vector of pcDNA3.1hisA-rnwas 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.%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pcDNA3.1 hisA,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-TGF31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.