| 2175 Pdgf Gene Delivery Sustains Gingival Fibroblast Signal Transduction | ||
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J. SUGAI1, Q. JIN2, and W. GIANNOBILE1, 1University of Michigan, Ann Arbor, USA, 2University of Michigan, School of Dentistry, Ann Arbor, USA OBJECTIVE: This study compared the effects of PDGF gene transfer versus PDGF protein delivery on gingival fibroblasts derived from periodontia to better understand the effects of PDGF on intracellular signal transduction pathways. METHODS: Primary cultures of human gingival fibroblasts were transduced for 3h with adenovirus encoding either human PDGF-B, PDGF-1308 (a dominant negative mutant of PDGF-A), or control vector (Ad-Luciferase), compared to 3h exposure with PDGF-BB (rhPDGF-BB). Cells were harvested at 6, 12, 24, 48, 72 and 96 h post-gene delivery and lysed for protein analysis and RNA. RT-PCR was performed to detect PDGF-B and GAPDH. Western blotting was performed to determine protein expression over time. A bead-based multiplex assay was utilized which simultaneously measures phosphorylation levels of six target cell signaling proteins (phospho-JNK, -MAPK, -Ik__a, -ERK1/2, -ATF2 and –Stat3) within the cell lysates. Finally, we examined cell signaling mediated by PDGF-B using a gene microarray system (Human EGF/PDGF Signaling Pathway Gene Array) on cDNA samples from above. RESULTS: Western blot analysis of PDGF-B receptor protein levels showed delayed, yet sustained expression in response to adenoviral delivery of PDGF-B compared to rhPDGF-BB treatment. Using the bead-based assay, it was found that c-jun N-terminal kinase (JNK), activating transcription factor-2 (ATF-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) follow a similar pattern of elevated, sustained phosphorylated protein expression in Ad-PDGF-B-treated HGF cells compared to either rhPDGF-BB or Ad-PDGF-1308 treatment. Finally, gene array analysis comparing Ad-PDGF-B treated HGF cells to rhPDGF-B-treated HGF cells, we noted differential gene expression patterns for multiple intracellular messages between the PDGF delivery approaches. CONCLUSION: These findings demonstrate that gene delivery of PDGF-B displays sustained signal transduction effects in human gingival fibroblasts above that of PDGF protein exposure. These data support the potential role of gene therapy to target cells derived from periodontia for tissue engineering applications. | ||
| Seq #239 - Wound Regeneration and Tissue Engineering 3:00 PM-4:00 PM, Saturday, 11 March 2006 Dolphin Hotel Pacific Hall | ||
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