| Introduction:Human dental apical papilla cells have mineralization potential, which play a key role in the root development of young permanent teeth. Limited literature is available about dental apical papilla cells mineralization in vitro and regeneration of dentin-like tissue in vivo on condition of gene transfection. In this study, the VEGF165gene was cloned and eucaryotic expression vector was constructed to investigate the effect of overexpressed VEGF165and TGFβ1on the mineral-related genes in human dental apical papilla cells and evaluate the regeneration of dentin-like tissue on the condition of VEGF165and TGFβ1proteins released from stably transfected Chinese hamster ovary cell (CHO)Methods:Total RNA of ECV304cell was extracted. The VEGF165gene 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-VEGF165and pcDNA3.1hisA-TGF(31were transfected into human dental apical papilla cells. Then the efficiency of gene transfection and the expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), dentin matrix protein1(DMP1) mRNAs were detected by real-time PCR. On the other hand, pcDNA3.1hisA-VEGF165and pcDNA3.1hisA-TGFβ1were seperately transfected in CHO. The cells stably expressing mRNAs and proteins were selected with G418sulfate and then were evaluated by RT-PCR for the VEGF,65and TGFβ1mRNAs. Besides, VEGF165and TGFβ1proteins in the supernatant were detected by ELISA. Then the cells were seeded on collagen membranes. The maxillary first molars on two sides of24wistar rats were selected as experimental teeth and the collagen membranes were seperately planted over the holes of artificial pulpal exposure. The cavities were filled with ChemFlex finally. The groups were divided randomly as following:(1) filled directly with ChemFlex;(2) collagen membranes included normal CHO+ChemFlex;(3) collagen membranes included CHO transfected with pcDNA3.1hisA+ChemFlex;(4) collagen membranes included CHO transfected with pcDNA3.1hisA-VEGF165+ChemFlex;(5) collagen membranes included CHO transfected with pcDNA3.1hisA-TGF(31+ChemFlex;(6) collagen membranes included CHO transfected with pcDNA3.1hisA-VEGF165and pcDNA3.1hisA-TGFpl+ChemFlex.8weeks later, the rats were killed and the specimens were treated following below procedures:fixed6d with10%neutral formalin, washed24h with running water, dehydrated by70%,90%,95%,100%alcohol, treated96h with chloroform, immersed with immerse water, embed with resin, sectioned by the machine of hard tissue slicing and dyed by toluidine blue. All statistical analyses were performed with SPSS17.0software. Differences were statistically significant when p<0.05.Results:Cloned VEGF165gene sequences inserted into expression vector showed100%homology related to the sequence in GenBank database. VEGF165and TGFβ1mRNA 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-TGFβ1and transfected with two plasmids (p<0.05). The expression of BSP mRNA were not varying (p>0.05), while no expression of DMP1mRNA exhibited in each experiment group. The results of RT-PCR and ELISA showed that CHO stably expressed VEGF165and TGFβ1mRNAs and proteins after transfection and selection. The result of toluidine blue staining showed that in the group of CHO transfected with pcDNA3.1hisA-VEGF165, the blood capillaries were congestive and inflammatory cells infiltrated obviously under the mechanical exposed pulpal site, but no hard tissue regenerated; in the group of CHO transfected with pcDNA3.1hisA-TGFβ1, a spot of colored mineralization pellets under the mechanical exposed pulpal site surrounded with hyperplastic fibroblast were observed, no tubular dentin and sporadic inflammatory cells were detected; in the group of CHO transfected with pcDNA3.1hisA-VEGF165and pcDNA3.1hisA-TGFβ1, generous colored mineralization pellets almostly closed the mechanical exposed pulpal site and columnar odontoblast were arranged orderly, no regular dentin bridge was detected; the hard tissue was not detected in the control group.Conclusions:The recombinant eucaryotic expression vector of pcDNA3.1hisA-VEGF165was constructed successfully. Overexpressed VEGF165and TGFβ1can induce the expression of most mineral-related genes and they may play a key role during the differentiation of human dental apical papilla cells. TGFβ1could promote the formation of mineralization pellets in vivo, and VEGF165and TGFβ1could promote the formation of mineralization pellets betterly. |
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