Objective To construct a replicationdefective recombinant adinovirus including the target gene human bone morphogenetic protein 4(fragment hBMP-4). Methods The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transformed into the packaging of thecell lines HEK293 to produce the replication-defective recombinant adenovirusescontaining the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay. Results The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by theWestern-blot assay. Conclusion The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
Objective To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor(VEGF) by different routes of administration. Methods Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carryingpcDNA3-VEGF121 (n=18), intramuscular injectionpcDNA3-VEGF121 (n=18), or pcDNA3 (as control group,n=14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 μg of the plasmid pcDNA 3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histologicalvessels of ischemic hindlimbs were measured respectively. Results In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographicscore and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when comparedwith the intramuscular-injection limbs. Conclusion These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121could augmentcollatal development and tissue perfusion in an animal model of hindlimb ischemia, andgelation sponge carrying VEGF gene may respect a potential therapy methods.