Fig. 1.
Adenoviral gene delivery of PDGF-B results in the sustained expression of platelet-derived growth factor-B in human gingival fibroblast cells. (A) Human gingival fibroblast cells were cultured to confluence and were then transferred to serum-free medium containing Ad-PDGF-B (multiplicity of infection = 200), Ad-Luc, Ad-PDGF-1308, or platelet-derived growth factor-BB protein (20 ng/mL). The cells were then allowed to incubate for 2–3 h before removal and replacement with serum-free medium. The cells were then harvested for RNA extraction at 6, 12, 24, 48, 72 and 96 h post-infection. Approximately 1.0 µg of total RNA was then used to generate cDNA with oligo dT primers. The resulting cDNA was used as a template for polymerase chain reaction amplification using human gene-specific primers for PDGF-B or GAPDH. The results show that only the treatment with Ad-PDGF-B caused a measurable increase in PDGF-B gene expression. The results obtained following treatment with Ad-Luc, Ad-PDGF-1308 or platelet-derived growth factor-BB protein showed no detectable expression of the PDGF-B gene (data not shown). (B) Approximately 2 × 105 cells were seeded in 100-cm dishes. After transient infection with Ad-PDGF-1308 or Ad-PDGF-B, the cells were cultured in serum-free medium. Conditioned media were collected at 96 h. After dialysis and lyophilization, samples were reconstituted in H2O, loaded for non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected using anti-PDGF-BB. Our Ad-PDGF-B vector encodes a 160 amino acid platelet-derived growth factor-B isoform with a molecular weight of 18.1 kDa; therefore, a dimer in the non-reduced SDS gel was about 36.2 kDa. For the recombinant human platelet-derived growth factor-BB control, the dimer of the 109 amino acid isoform displayed a molecular weight of 25 kDa. hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; PDGF-B, platelet-derived growth factor-B; rhPDGF-BB, recombinant human platelet-derived growth factor-BB.