OBJECTIVETo clone the VEGF165 gene and to construct eucaryotic expression vector, investigate the effect of overexpressed VEGF165 and transforming growth factor beta1 (TGFbeta1) on the mineral-related genes in human cells from apical papilla.

METHODSTotal 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-TGFbeta1 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).

RESULTSCloned VEGF165 gene sequences and inserted into expression vector of the VEGF165 sequences showed 100% homology related to the sequence in GenBank database. VEGF165 and TGFbeta1 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-TGFbeta1 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 eucaryotic expression vector of pcDNA3.1hisA-VEGF165 was constructed successfully. VEGF165 and TGFbeta1 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.