Experimental Study On The Proliferation Of Epidermal Stem Cells Transfected Of Hpdgf-bb Mediated By Adenovirus In Vitro

Objectives:(1) To construct a recombinant adenovirus vector (rad-PDGF)containing PDGF-BB gene.(2) To observe the effects of proliferation of hESCs after Transfection by rad-PDGF.Methods:(1) hPDGF-BB was amplified by PCR and cloned into the transfer vector pAd-Track-CMV to construct a transfer plasmid pAd-Track-CMV-PDGF-BB. The plasmid cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 to achieve homologous recombination (rad-PDGF). After being digested with Pac I, the DNA of identified recombinant plasmid was transfected into HEK293 cells with lipofectaminTM 2000 to package and amplify adenovirus. After the viruses were harvested, the infectious titer of the recombinant adenovirus was tested, and the PCR products of the viruses ware analyzed.(2) hESCs were separated from the human prepuce, cultured in vitro, and identified, and infected with the recombinant adenoviruses. The expression of green fluorescent protein in hESCs modificated by rad-PDGF was visualized in fluorescence microscope. The expression of the target gene was detected by Western blotting. Its effect on the proliferation of the infected hESCs was examined by MTT.Results:(1) PDGF-BB cDNA had been linked with pAd-Track-CMV vector with correct order. The result of DNA sequencing showed that the sequence of gene combined with pAd-Track-CMV-PDGF-BB was identical to the coding region of PDGF-BB gene in Genebank. Homologous recombination had completed successfully between pAd-Track-CMV-PDGF-BB and adenoviral backbone plasmid pAdEasy-1. The recombinant adenoviral plasmid could be digested with Pac I to produce a fragment whose size was 4.5kb. The infectious titer of adenovirus was 1×109pfu/ml. the size of PCR products is consistent with the size of the target gene.(2) hESCs strongerly expressed the K19. The percentage of putative hESCs infected by rad-PDGF which showed expression of GFP was about 85%. rad-PDGF can expressed PDGF-BB in infected hESCs. The proliferation of hESCs transfected with the recombinant virus was significantly increased in comparison with those of the control hESCs(P<0.05).Conclusion:(1) The rad-PDGF was successfully constructed;(2) rad-PDGF was transfected into hESCs and the PDGF-BB protein was expressed in infected hESCs successfully. hPDGF-BB can increase the growth of hESCs.