重组腺病毒介导的人表皮生长因子基因体外转染人牙髓干细胞的分析.pdfVIP

vitro and digested the recombinant adenovirus vector pAd-EGF with enzyme XbaI. Transfected the cell HEK293 with linearizated recombinant adenovirus vector and packaged to reconstructed adenovirus rAd-EGF which was verified by PCR and measured the viral titer. The human dental pulp stem cells which during exponential phase of growth were divided into 3 groups 24 hours before infected ：Group1 ，infected with recombinant adenovirus rAd-EGF ；Group 2 ，the control group, infected with adenovirus rAd-NC ；Group 3 ，the blank. Added 100 MOI recombinant adenovirus rAd -EGF or adenovirus rAd-NC into group1 and group 2,add the same amount DMEM into group 3 when the cell density reach to 70%,cultured at 37℃,5%C02 for 4 hours. Replaced with fresh complete medium and cultured for another 48 hours. Digestion with Trypsin to collect the cells in 48 hours, use Western-blot experiments to detect different EGF protein expression in each group after gene transfect ion. Results: Use microscopic to observe the attachment and growth of dental pulp stem cells, the cell is spindle or polygonal, each cell has 2 or 3 processes and growth well. KpnI, XhoI site restriction endonuclease analysis showed that the adenovirus shuttle vector pYr-ads-1-EGF was successfully constructed. Restriction enzyme digestion and DNA sequencing demonstrated successful construction of adenovirus vector pAd-EGF. Identification by PCR shows that the recombinant adenovirus of rAd-EGF packaging success. Western-blot analysis EGF protein expression on each group after recombinant adenovirus rAd-EGF infected hDPSCs in 48 hours. The results show that the infection group of EGF protein levels were significantly higher than that of other two groups