Research On Adenovirus Mediated Human Epidermal Growth Factor Gene Transfect Human Dental Pulp Stem Cell In Vitro

Objective: This experiment is to culture the human dental pulp stem cellsto the third generation of the exponential growth phase in vitro. Thensubcloned the coding sequence fragment of EGF gene into the adenovirusshuttle vector pYr-adshuttle-1and reconstructed it with adenovirus vectorpAd/BL-DEST in vitro. Transfect pAd/BL-DEST to HEK293cell．Afterrecombinant adenovirus vector was successfully constructed, transfectedit to the exponential phase of dental pulp stem cells and explore the EGFprotein expression of human dental pulp stem cells, and to providematerials and data for subsequent experiments.Methods: Subcultured the human dental pulp stem cells to the thirdgeneration with DEME medium containing10%FBS in vitro, observedthe attachment and growth of hDPSCs under microscope. Doubledigested pCR4-TOPO-EGF and adenovirus shuttle vectorpYr-adshuttle-1,recovered the fragment of EGF and linear adenovirusshuttle vector respectively by agarose gel electrophoresis with aconnection, then transferred it to competent E.coli DH5α to amplify forcollecting recombinant plasmid pYr-adshuttle-1-EGF. Authenticate thisplasmid by restriction enzyme digestion. Subcoloned iNOS expressioncassette to adenovirus express vector pAd/BL-DEST by LR reaction in vitro and digested the recombinant adenovirus vector pAd-EGF withenzyme XbaI. Transfected the cell HEK293with linearizatedrecombinant adenovirus vector and packaged to reconstructed adenovirusrAd-EGF which was verified by PCR and measured the viral titer. Thehuman dental pulp stem cells which during exponential phase of growthwere divided into3groups24hours before infected：Group1，infectedwith recombinant adenovirus rAd-EGF；Group2，the control group,infected with adenovirus rAd-NC；Group3，the blank. Added100MOIrecombinant adenovirus rAd-EGF or adenovirus rAd-NC into group1andgroup2,add the same amount DMEM into group3when the cell densityreach to70%,cultured at37℃,5%C02for4hours. Replaced with freshcomplete medium and cultured for another48hours. Digestion withTrypsin to collect the cells in48hours, use Western-blot experiments todetect different EGF protein expression in each group after gene transfection.Results: Use microscopic to observe the attachment and growth of dentalpulp stem cells, the cell is spindle or polygonal, each cell has2or3processes and growth well. KpnI, XhoI site restriction endonucleaseanalysis showed that the adenovirus shuttle vector pYr-ads-1-EGF wassuccessfully constructed. Restriction enzyme digestion and DNAsequencing demonstrated successful construction of adenovirus vectorpAd-EGF. Identification by PCR shows that the recombinant adenovirus of rAd-EGF packaging success. Western-blot analysis EGF proteinexpression on each group after recombinant adenovirus rAd-EGF infectedhDPSCs in48hours. The results show that the infection group of EGFprotein levels were significantly higher than that of other twogroups(P<0.05).Conclusions: Cultured hDPSCs to third generation, the cells grew welland proliferated normally. Restriction enzyme digestion demonstratedsuccessful construction of adenovirus shuttle vector pYr-adshuttle-1-EGFand adenovirus vector pAd-EGF. Adenovirus packaging for HEK293cells to obtain recombinant adenovirus of rAd-the EGF. Transfected therecombinant adenovirus of rAd-EGF to human dental pulp stem cells, andthe expression of EGF protein increased. Adenovirus is an efficienttransfection vector, the EGF gene can be effectively transfected to humandental pulp stem cells by adenovirus-mediated.