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