Abstract:

Objective　To construct the recombinant adenovirus vector containing PDGF-BB gene and detect its expression in human epidermal stem cells (hESCs).　Methods　The core sequence of human PDGF-BB was amplified by PCR from pCMV-SPORT6, and then was cloned to pAd track-CMV. The linearized shuttle plasmid was homogenously recombined with pAdeasy-l in BJ5183, and the potential clone was analyzed by restriction endonuclease digestion. The correct clone was linearized and transfected into HEK293 cells for packing and amplifying so as to obtain adenovirus rAD-PDGF. hESCs were separated from the human prepuce, cultured in vitro, and identified by flow cytometry. hESCs were infected by the harvested virus, and the protein expression of PDGF-BB in hESCs was respectively detected by Western blot.　Results　PDGF-BB was correctly cloned into the adenovirus vector, and the percentage of putative epidermal stem cells showed expression of the molecular marker was about 88%. The recombinant adenovirus vector containing human PDGF-BB gene was constructed successfully, which could infect hESCs efficiently, with the expression of the PDGF-BB in the infected hESCs detected by Western blotting.　Conclusions　 The human ESCs infected by constructed adenovirus vector rAD-PDGF could express PDGF-BB successfully.

Key words: Platelet-derived growth factor (PDGE-BB), Adenovirus, Human epidermal stem cells(hESCs)