Experimental Study Of Gene Transfer To Reverse The Disc Degeneration

Objective: To provide a highly infective adenoviral vector Ad/CMV-hTGFβ1 for the gene therapy of intervertebral disc degeneration.Methods: The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid is linerized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally the linerized recombinant plasmid is transfected into adenovirus packaging cell lines(293 cells). Recombinant adenoviruses are generated within 2 weeks.Results: The recombinant adenoviral plasmid was cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electroporesis. The infected 293 cells showed evident CPE(cytopathic effect). The resultants of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFβ1 was verified by immunohistochemical staining.Conclusion: The successful construction of the adenoviral vector Ad/CMV- hTGFβ1 made it possible for the following study of gene therapy for intervertebral disc degeneration.